Vector

ABSTRACT

A vector comprising a nucleotide sequence of interest (“NOI”) encoding a product of interest (“POI”) is described. The NOI and/or the POI is capable of recognizing a tumor, such that in use the vector is capable of delivering the NOI and/or the POI to the tumor.

This application is a continuation of U.S. patent application Ser. No.11/380,188, filed Apr. 25, 2006, which is a continuation of U.S. patentapplication Ser. No. 10/104,522, filed Mar. 22, 2002, which is adivisional of U.S. patent application Ser. No. 09/445,375, filed Mar.21, 2000, which is the U.S. national phase of International ApplicationNo. PCT/GB98/01627, which claims the benefit of priority of UnitedKingdom Patent Applications Nos. 9711579.4, 9713150.2 and 9714230.1,filed on Jun. 4, 1997, Jun. 20, 1997 and Jul. 4, 1997, respectively.

The present invention relates to a vector, preferably for use inmedicine.

As it is well known in the art, a vector is a tool that allows orfaciliates the transfer of an entity from one environment to another. Byway of example, some vectors used in recombinant DNA techniques allowentities—such as a segment of DNA (such as a heterologous DNA segment,such as a heterologous cDNA segment)—to be transferred into a targetcell. Optionally, once within the target cell, the vector may then serveto maintain the heterologous DNA within the cell or may act as a unit ofDNA replication. Examples of vectors used in recombinant DNA techniquesinclude plasmids, chromosomes, artificial chromosomes or viruses.

Thus, vectors can be used to deliver proteins and/or nucleotidesequences to targeted cells, such as tumour cells.

However, as it is well known, nucleotide sequences and proteins arecomplex molecules which may be produced from biological sources, mostusually from genetically engineered organisms or cell cultures.Furthermore, the procedures for the production of nucleotide sequencesand proteins can be complicated, labour intensive and costly.Furthermore, pharmacological properties and other aspects of thefunction of some proteins—such as immunoglobulins derived from non-humanbiological sources—and nucleotide sequences may frequently differ inimportant ways from the activity of the corresponding natural humanimmunoglobulins produced in human cells. By way of backgroundinformation, an immunoglobulin is a member of a family of relatedmultimeric proteins which are normally secreted from cells of theB-lymphocyte lineage of a vertebrate, whose typical function is to bindspecifically with a region of a macromolecule identified as non-self.Immunoglobulins represent a major component of the immune responserepertoire of the organism and are synonymous with“antibodies”. Onemajor cause of such differences in activity may be due to variations inthe pattern of glycosylation of proteins derived from different species(reviewed in Bebbington 1995; In Monoclonal Antibodies: the secondgeneration ed. H. Zola pg 165-181). Furthermore, systemic administrationof proteins (especially those containing toxin domains) and nucleotidesequences can induce additional pharmacokinetic and toxicologicalproblems (reviewed in Scheinberg and Chapman 1995. In Monoclonalantibodies (ed. Birch and Lennox) Chapter 2.1).

Thus, the present invention seeks to provide an improved vector systemfor delivering a nucleotide sequence of interest and/or a productexpressed by the same.

According to a first aspect of the present invention there is provided avector comprising a nucleotide sequence (“NS”) coding for a tumourinteracting protein (“TIP”) and optionally comprising a nucleotidesequence of interest (“NOI”) which NOI encodes a product of interest(“POI”); wherein the TIP is capable of recognising a tumour, such thatin use the vector is capable of delivering the NOI and/or the POI to thetumour.

According to a second aspect of the present invention there is provideda method of delivering a nucleotide sequence of interest (“NOI”) and/ora product of interest (“POI”) encoded by same to a tumour, wherein theNOI and/or POI are delivered to the tumour by use of a vector comprisingthe NOI and/or expressing the POI; wherein the NOI and/or the POI iscapable of recognising a tumour; wherein the NOI and/or the POI isdelivered to the tumour; and wherein the vector is a vector according tothe present invention.

According to a third aspect of the present invention there is providedthe use of a vector to deliver a nucleotide sequence of interest (“NOI”)and/or a product of interest (“POI”) encoded by same to a tumour,wherein the NOI and/or POI are delivered to the tumour by use of thevector which comprises the NOI and/or expresses the POI; wherein the NOIand/or the POI is capable of recognising a tumour when the NOI and/orthe POI is delivered to the tumour; and wherein the vector is a vectoraccording to the present invention.

According to a fourth aspect of the present invention there is provideda method of treating a subject in need of same, the method comprisingdelivering a nucleotide sequence of interest (“NOI”) and/or a product ofinterest (“POI”) encoded by same to a tumour, wherein the NOI and/or POIare delivered to the tumour by use of a vector comprising the NOI and/orexpressing the POI; wherein the NOI and/or the POI is capable ofrecognising a tumour; wherein the NOI and/or the POI is delivered to thetumour; and wherein the vector is a vector according to the presentinvention.

According to a fifth aspect of the present invention there is providedthe use of a genetic vectors to deliver a therapeutic gene encoding aTIP—preferably a tumour binding protein (“TBP”)—more preferably asecretable TIP (preferably a secretable TBP)—to the interior of a tumourmass.

According to a sixth aspect of the present invention there is provided agene delivery system for targeting one or more genes encoding a TIP(preferably a TBP) to a tumour, comprising a genetic vector encoding aTIP (preferably a TBP) and an in vivo gene-delivery system.

According to a seventh aspect of the present invention there is provideda method of treating cancer comprising administering a TIP (preferably aTBP) gene or genes in a gene delivery system according to the presentinvention either systemically or directly to the site of a tumour.

According to an eighth aspect of the present invention there is provideda gene delivery system for introducing one or more genes encoding a TIP(preferably a TBP) into cells of the haematopoietic (preferably myeloidhaematopoietic) cell lineage either in vivo or ex vivo.

According to a ninth aspect of the present invention there is provided amethod for treating cancer in a mammal, comprising administering to anindividual a gene delivery system according to the present inventionthat is capable of expressing a TBP in cells derived from ahaematopoietic (preferably myeloid haematopoietic) origin.

According to a tenth aspect of the present invention there is provided agenetic vector comprising a therapeutic gene or genes encoding a TIP(preferably a TBP), operably linked to an expression regulatory elementselectively functional in a cell type present within a tumour mass.

According to an eleventh aspect of the present invention there isprovided a genetic vector comprising a therapeutic gene or genes isdelivered to the interior of the tumour wherein the therapeutic geneencodes a TIP (preferably a TBP), which additionally contains one ormore effector domains.

According to a twelfth aspect of the present invention there is provideda method of treating cancer in a mammal which comprises administering toan individual a combination of a cytokine or a cytokine-encoding geneand one or more TIP (preferably a TBP) genes according to any of theprevious aspects of the invention.

According to a thirteenth aspect of the present invention there isprovided the delivery of TIP—(preferably a TBP-) encoding genes to thesite of a tumour.

Preferably the vector comprises the NOI.

In one preferred aspect, the vector is expressing the POI.

The vector of the present invention may be useful for inter alia medicalapplications—such as diagnostic or therapeutic applications.

Preferably the NOI is a therapeutic NOI and/or the POI is a therapeuticPOI.

On occasions in the following text, the NS and NOI may be individuallyor collectively referred to as being a gene.

The NS and NOI can be any suitable nucleotide sequence. For example,independently they can be DNA or RNA—which may synthetically prepared ormay be prepared by use of recombinant DNA techniques or may be isolatedfrom natural sources or may be combinations thereof. The NOI may be asense sequence or an antisense sequence.

There may be a plurality of NSs or NOIs, which may be directly orindirectly joined to each other, or combinations thereof. Thus, theexpressed product may have two or more effector domains (which may bethe same or different) and/or two or more TIP domains (which may be thesame or different).

Preferably in use the vector is capable of delivering the NOI and/or thePOI to the interior of a tumour mass.

In addition to cancerous cell, the cell types present within a tumourmass include but are not limited to macrophages, lymphocytes, tumourinfiltrating lymphocyes, endothelial cells etc.

Preferably the NS and/or the TIP comprises at least one tumour bindingdomain capable of interacting with at least one tumour associated cellsurface molecule (“TACSM”).

In accordance with the present invention the TACSM can include but isnot limited to a cell surface molecule which plays a role in tumour cellgrowth, migration or metastasis, a receptor for adhesive proteins suchas the integrin vitronectin receptor, a growth factor receptor (such asepidermal growth factor (EGF) receptor, platelet-derived growth factor(PDGF) receptor, fibroblast-derived growth factor (FDGF) receptor, nervegrowth factor receptor, insulin-like growth factor (IGF-1) receptor; aplasminogen activator; a metalloproteinase (such as colllagenase) 5T4antigen; a tumour specific carbohydrate moiety; an oncofetal antigen; amucin; a growth factor receptor; a glycoprotein; and an antigenrestricted in its tissue distribution.

Preferably the TACSM is selectively expressed on one cell type or on arestrictive number of cell types.

Preferably in use the vector is capable of delivering the NOI and/or thePOI to a selective tumour site.

Preferably the TIP is or comprises a tomour binding protein (“TBP”).

Preferably the TIP is a TBP.

Examples of a TBP include: an adhesion molecule such as Intercellularadhesion molecule, ICAM-1, ICAM-2, LFA-1, LFA-2, LFA-3, LECAM-1, VLA-4,ELAM, N-CAM, N-cadherin, P-Selectin, CD44 and its variant isoforms (inparticular CD44v6, CD44v7-8), CD56; a growth factor receptor ligand suchepidermal growth factor (EGF), Platelet-derived growth factor (PDGF),Fibroblast-derived growth factor (FDGF), Nerve growth factor,vasopressin, insulin, insulin-like growth factor (IGF-1), hepatocytegrowth factor, nerve growth factor, human growth factor, brain derivedgrowth factor, ciliary neutrophic factor, glial cell line-derived growthfactor; heavy and light chain sequences from an immunoglobulin (Ig)variable region (from human and animal sources), engineered antibody orone from a phage display library. A phage display library is a techniqueof expressing immunoglobulin genes in bacteriophage has been developedas a means for obtaining antibodies with the desired bindingspecificities. Expression systems, based on bacteriophage lambda, andmore recently filamentous phage have been developed. The bacteriophageexpression systems can be designed to allow heavy and light chains toform random combinations which are tested for their ability to bind thedesired antigen.

The TBP may contain an effector domain which is activated on binding ofthe TPB to the TASCM. The effector domain or momains may be activated onbinding of the TBP to a TASCM leading to inhibition of tumour cellproliferation, survival or dissemination. The effector domain maypossess enzymatic activity (such as a pro-drug activating enzyme) or theeffector domain may include a toxin, or an immune enhancer, such as acytokine/lymphokine such as those listed above.

Preferably the TBP comprises one or more binding domains capable ofinteracting with one or more TACSMs which are present on the cancerouscells—which TACSMs may be the same or different.

The term “interacting” includes direct binding, leading to a biologicaleffect as a result of such binding.

Preferably the TIP is or comprises at least part of an antibody.

As is well known, antibodies play a key role in the immune system. Inbrief, the immune system works in three fundamentally different ways: byhumoral immunity, by cellular immunity and by secretion of stimulatoryproteins, called lymphokines Humoral immunity relies on proteinscollectively called immunoglobulin which constitute about 20% of theproteins in the blood. A singly immunoglobulin molecule is called anantibody but “antibody” is also used to mean many different moleculesall directed against the same target molecule. Humoral immunity alsoinvolves complement, a set of proteins that are activated to killbacteria both nonspecifically and in conjunction with antibody.

In cellular immunity, intact cells are responsible for recognition andelimination reactions. The body's first line of defense is therecognition and killing of microorganisms by phagocytes, cellsspecialised for the ingestion and digestion of unwanted material. Thesecells include neutrophils and macrophages. A key role of antibodies isto help phagocytes recognise and destroy foreign materials.

In order to perform these functions, the antibody is divided into tworegions: binding (Fab) domains that interact with the antigen andeffector (Fc) domains that signal the initiation of processes such asphagocytosis. Each antibody molecule consists of two classes ofpolypeptide chains, light (L) chains and heavy (H) chains. A singleantibody has two identical copies of the L chain and two of the H chain.The N-terminal domain from each chain forms the variable regions, whichconstitute the antigen-binding sites. The C-terminal domain is calledthe constant region. The variable domains of the H (V_(H)) and L (V_(L))chains constitute an Fv unit and can interact closely to form a singlechain Fv (ScFv) unit. In most H chains, a hinge region is found. Thishinge region is flexible and allows the Fab binding regions to movefreely relative to the rest of the molecule. The hinge region is alsothe place on the molecule most susceptible to the action of proteasewhich can split the antibody into the antigen binding site (Fab) and theeffector (Fc) region.

The domain structure of the antibody molecule is favourable to proteinengineering, facilitating the exchange between molecules of functionaldomains carrying antigen-binding activities (Fabs and Fvs) or effectorfunctions (Fc). The structure of the antibody also makes it easy toproduce antibodies with an antigen recognition capacity joined tomolecules such as toxins, lymphocytes or growth factors.

Monoclonal antibodies are homogenous antibodies of the same antigenicspecificity representing the product of a single clone ofantibody-producing cells. It was recognised that monoclonal antibodiesoffered the basis for human therapeutic products. However, althoughmouse antibodies are similar to human antibodies, they are sufficientlydifferent that they are recognised by the immune system as foreignbodies, thereby giving rise to an immunological response. Thishuman-anti-mouse-antibody (HAMA) response limits the usefulness of mouseantibodies as human therapeutic products.

Chimeric antibody technology involves the transplantation of whole mouseantibody variable domains onto human antibody constant domains. Chimericantibodies are less immunogenic than mouse antibodies but they retaintheir antibody specificity and show reduced HAMA responses.

In chimeric antibodies, the variable region remains completely murine.However, the structure of the antibody makes it possible to producevariable regions of comparable specificity which are predominantly humanin origin. The antigen-combining site of an antibody is formed from thesix complementarity-determining regions (CDRs) of the variable portionof the heavy and light chains. Each antibody domain consists of sevenantiparallel β-sheets forming a β-barrel with loops connecting theβ-strands. Among the loops are the CDR regions. It is feasible to morethe CDRs and their associated specificity from one scaffolding β-barrelto another. This is called CDR-grafting. CDR-grafted antibodies appearin early clinical studies not to be as strongly immunogenic as eithermouse or chimeric antibodies. Moreover, mutations may be made outsidethe CDR in order to increase the binding activity thereof, as inso-called humanised antibodies.

Fab, Fv, and single chain Fv (ScFv) fragments with VH and VL joined by apolypeptide linker exhibit specificities and affinities for antigensimilar to the original monoclonal antibodies. The ScFv fusion proteinscan be produced with a nonantibody molecule attached to either the aminoor carboxy terminus. In these molecules, the Fv can be used for specifictargeting of the attached molecule to a cell expressing the appropriateantigen. Bifunctional antibodies can also be created by engineering twodifferent binding specificities into a single antibody chain.Bifunctional Fab, Fv and ScFv antibodies may comprise engineered domainssuch as CDR grafted or humanised domains.

In virally directed enzyme therapy (VDEPT), a foreign gene is deliveredto normal and cancerous cells by a viral vector—such as a retroviralvector. The foreign gene codes for an enzyme that can convert anon-toxic prodrug (e.g. 5-fluorocytosine) to a toxic metabolite(5-fluorouracil) that will kill those cells making it (Sikora et at 1994Ann New York Acad Sci 71b: 115-124). If the promoter utilised is tumourspecific, then the toxic product will only be synthesised in the tumourcells. Studies in animal models have demonstrated that this type oftreatment can deliver up to 50-fold more drug than by conventional means(Connors and Knox 1995 1995 Stem Cells 13: 501-511). A variation of thistechnique uses tumour associated antibodies conjugated to prodrugconverting enzymes to provide specific delivery to tumours. This methodis referred to as antibody-directed enzyme prodrug therapy (ADEPT)(Maulik S and Patel S D “Molecular Biotechnology”1997 Wiley-Liss Inc. pp45).

A large number of monoclonal antibodies and immunoglobulin-likemolecules are known which bind specifically to antigens present on thesurfaces of particular cell types such as tumour cells. Procedures foridentifying, characterising, cloning and engineering these molecules arewell established, for example using hybridomas derived from mice ortransgenic mice, phage-display libraries or scFv libraries. Genesencoding immunoglobulins or immunoglobulin-like molecules can beexpressed in a variety of heterologous expression systems. Largeglycosylated proteins including immunoglobulins are efficiently secretedand assembled from eukaryotic cells, particularly mammalian cells.Small, non-glycosylated fragments such as Fab, Fv, or scFv fragments canbe produced in functional form in mammalian cells or bacterial cells.

The immunoglobulin or immunoglobulin-like molecule may be derived from ahuman antibody or an engineered, humanised rodent antibody such as aCDR-grafted antibody or may be derived from a phage-display library ormay be a synthetic immunoglobulin-like molecule.

The antigen-binding domain may be comprised of the heavy and lightchains of an immunoglobulin, expressed from separate genes, or may usethe light chain of an immunoglobulin and a truncated heavy chain to forma Fab or a F(ab)₂ fragment. Alternatively, truncated forms of both heavyand light chains may be used which assemble to form a Fv fragment. Anengineered scFv fragment may also be used, in which case, only a singlegene is required to encode the antigen-binding domain. In one preferredaspect, the antigen-binding domain is formed from a Fv or a scFv.

When a pathogen invades the body, lymphocytes respond with three typesof reaction. The lymphocytes of the humoral system (B cells) secreteantibodies that can bind to the pathogen, signalling its degradation bymacrophages and other cells. The lymphocytes of the cellular system (Tcells) carry out two major types of functions. Cytotoxic T lymphocytes(CTLs) develop the ability to directly recognise and kill the cellsinfected by the pathogen. Helper T cells (TH cells) independentlyrecognise the pathogen and secrete protein factors (lymphokines) thatstimulate growth and responsiveness of B cells, T cells, andmacrophages, thus greatly strengthening the power of the immuneresponse.

Thus, in one preferred aspect, the TIP comprises an immunoglobulin, or apart thereof, or a bioisostere thereof.

In a preferred embodiment, the TIP comprises IgG and/or IgE, or a partthereof, or a bioisostere thereof.

In a more preferred embodiment, the TIP comprises IgE, or a partthereof, or a bioisostere thereof.

Preferably the TIP recognises a trophoblast cell surface antigen.

Preferably the TIP recognises the 5T4 antigen.

The trophoblast cell surface antigen, originally defined by monoclonalantibody 5T4 (Hole and Stern 1988 Br. J. Cancer 57; 239-246), isexpressed at high levels on the cells of a wide variety of humancarcinomas (Myers et al. 1994 J. Biol. Chem. 269; 9319-9324) but, innormal tissues of non-pregnant individuals, is essentially restricted tolow level expression on a few specialised epithelia (Myers et al. ibid.and references therein). The 5T4 antigen has been implicated incontributing to the development of metastatic potential and thereforeantibodies specifically recognising this molecule may have clinicalrelevance in the treatment of tumours expressing the antigen.

The variable region of the 5T4 monoclonal antibody can also be humanisedby a number of techniques, which are known in the art, includinggrafting of the CDR region sequences on to a human backbone. These canthen be used to construct an intact humanised antibody or a humanisedsingle chain antibody (Sab), such as an ScFv coupled to an Fc region(see Antibody Engineering: a practical approach, ed McCafferty et al.1996 OUP).

Here the term Sab is not limited to just a human or a humanised singlechain antibody. Preferably, however the Sab is a human single chainantibody or a humanised single chain antibody, or part thereof—such asScFv coupled to an Fc region.

Preferably the NS and NOI and/or the TIP and POI are linked together.

Preferably the TIP and POI are directly linked together.

Preferably any one or more of the NS, NOI, TIP, and POI further compriseat least one additional functional component.

Preferably, at least the TIP and/or POI further comprise at least oneadditional functional component.

Preferably the additional functional component is selected from any oneor more of a signalling entity (such as a signal peptide), an immuneenhancer, a toxin, or a biologically active enzyme.

In a preferred aspect the POI is a secretable POI. Thus, in this aspectof the present invention, preferably, the additional functionalcomponent is at least an entity capable of causing the POI to besecreted—such as a signalling entity.

Another preferred additional component is a promoter.

The term “promoter” is used in the normal sense of the art, e.g. an RNApolymerase binding site in the Jacob-Monod theory of gene expression.

Preferably the vector comprises a tumour specific promoter enhancer.

Other preferred additional components include entities enablingefficient expression of the POI. For example, the additional componentmay be an enhancer. Here, the term enhancer includes a DNA sequencewhich binds to other protein components of the transcription initiationcomplex and thus facilitates the initiation of transcription directed byits associated promoter.

Preferably the vector is used to deliver the NOI and/or POI ex vivoand/or in vivo to the tumour.

The vector of the present invention is useful in gene therapy fordelivering the NOI and/or the POI to a selective site.

Gene therapy includes any one or more of: the addition, the replacement,the deletion, the supplementation, the manipulation etc. of one or morenucleotide sequences in, for example, one or more targeted sites—such astargeted cells. If the targeted sites are targeted cells, then the cellsmay be part of a tissue or an organ. General teachings on gene therapymay be found in Molecular Biology (Ed Robert Meyers, Pub VCH, such aspages 556-558).

By way of further example, gene therapy also provides a means by whichany one or more of: a nucleotide sequence, such as a gene, can beapplied to replace or supplement a defective gene; a pathogenic gene orgene product can be eliminated; a new gene can be added in order, forexample, to create a more favourable phenotype; cells can be manipulatedat the molecular level to treat cancer (Schmidt-Wolf and Schmidt-Wolf,1994, Annals of Hematology 69; 273-279) or other conditions—such asimmune, cardiovascular, neurological, inflammatory or infectiousdisorders; antigens can be manipulated and/or introduced to elicit animmune response—such as genetic vaccination.

The vector of the present invention may be a viral vector or a non-viralvector. Non-viral delivery systems include but are not limited to DNAtransfection methods. Here transfection includes a process using anon-viral vector to deliver a gene to a target mammalian cell. Typicaltransfection methods include electroporation, DNA biolistics,lipid-mediated transfection, compacted DNA-mediated transfection,liposomes, immunoliposomes, lipofectin, cationic agent-mediated,cationic facial amphiphiles (CFAs) (Nature Biotechnology 1996 14; 556),and combinations thereof. Viral delivery systems include but are notlimited to adenovirus vector, an adeno-associated viral (AAV) vector, aherpes viral vector, retroviral vector, lentiviral vector, baculoviralvector. Other examples of vectors include ex vivo delivery systems—whichinclude but are not limited to DNA transfection methods such aselectroporation, DNA biolistics, lipid-mediated transfection, compactedDNA-mediated transfection).

Preferably the vector is a viral vector.

Preferably the vector is a retroviral vector.

In recent years, retroviruses have been proposed for use in genetherapy. Essentially, retroviruses are RNA viruses with a life cycledifferent to that of lytic viruses. In this regard, when a retrovirusinfects a cell, its genome is converted to a DNA form. In slightly moredetail, a retrovirus is a virus which contains genomic RNA which onentry into a host cell is converted to a DNA molecule by a reversetranscriptase enzyme. The DNA copy serves as a template for theproduction of new RNA genomes and virally encoded proteins necessary forthe assembly of infectious viral particles. Thus, a retrovirus is aninfectious entity that replicates through a DNA intermediate.

There are many retroviruses and examples include: murine leukemia virus(MLV), human immunodeficiency virus (HIV), equine infectious anaemiavirus (EIAV), mouse mammary tumour virus (MMTV), Rous sarcoma virus(RSV), Fujinami sarcoma virus (FuSV), Moloney murine leukemia virus(Mo-MLV), FBR murine osteosarcoma virus (FBR MSV), Moloney murinesarcoma virus (Mo-MSV), Abelson murine leukemia virus (A-MLV), Avianmyelocytomatosis virus-29 (MC29), and Avian erythroblastosis virus(AEV).

A detailed list of retroviruses may be found in Coffin et at(“Retroviruses”1997 Cold Spring Harbour Laboratory Press Eds: J MCoffin, S M Hughes, H E Varmus pp 758-763).

Details on the genomic structure of some retroviruses may be found inthe art. By way of example, details on HIV may be found from the NCBIGenbank (i.e. Genome Accession No. AF033819).

All retroviruses contain three major coding domains, gag, pol, env,which code for essential virion proteins. Nevertheless, retroviruses maybe broadly divided into two categories: namely, “simple” and “complex”.These categories are distinguishable by the organisation of theirgenomes. Simple retroviruses usually carry only this elementaryinformation. In contrast, complex retroviruses also code for additionalregulatory proteins derived from multiple spliced messages.

Retroviruses may even be further divided into seven groups. Five ofthese groups represent retroviruses with oncogenic potential. Theremaining two groups are the lentiviruses and the spumaviruses. A reviewof these retroviruses is presented in “Retroviruses” (1997 Cold SpringHarbour Laboratory Press Eds: J M Coffin, S M Hughes, H E Varmus pp1-25).

All oncogenic members except the human T-cell leukemia virus-bovineleukemia virus group (HTLV-BLV) are simple retroviruses. HTLV, BLV andthe lentiviruses and spumaviruses are complex. Some of the best studiedoncogenic retroviruses are Rous sarcoma virus (RSV), mouse mammarytumour virus (MMTV) and murine leukemia virus (MLV) and the human T-cellleukemia virus (HTLV).

The lentivirus group can be split even further into “primate” and“non-primate”. Examples of primate lentiviruses include the humanimmunodeficiency virus (HIV), the causative agent of humanauto-immunodeficiency syndrome (AIDS), and the simian immunodeficiencyvirus (SIV). The non-primate lentiviral group includes the prototype“slow virus” visna/maedi virus (VMV), as well as the related caprinearthritis-encephalitis virus (CAEV), equine infectious anaemia virus(EIAV) and the more recently described feline immunodeficiencey virus(FIV) and bovine immunodeficiencey virus (BIV).

A distinction between the lentivirus family and other types ofretroviruses is that lentiviruses have the capability to infect bothdividing and non-dividing cells (Lewis et at 1992 EMBO. J 11; 3053-3058,Lewis and Emerman 1994 J. Virol. 68: 510-516). In contrast, otherretroviruses—such as MLV—are unable to infect non-dividing cells such asthose that make up, for example, muscle, brain, lung and liver tissue.

During the process of infection, a retrovirus initially attaches to aspecific cell surface receptor. On entry into the susceptible host cell,the retroviral RNA genome is then copied to DNA by the virally encodedreverse transcriptase which is carried inside the parent virus. This DNAis transported to the host cell nucleus where it subsequently integratesinto the host genome. At this stage, it is typically referred to as theprovirus. The provirus is stable in the host chromosome during celldivision and is transcribed like other cellular proteins. The provirusencodes the proteins and packaging machinery required to make morevirus, which can leave the cell by a process sometimes called“budding”.

As already indicated, each retroviral genome comprises genes called gag,pol and env which code for virion proteins and enzymes. These genes areflanked at both ends by regions called long terminal repeats (LTRs). TheLTRs are responsible for proviral integration, and transcription. Theyalso serve as enhancer-promoter sequences. In other words, the LTRs cancontrol the expression of the viral gene. Encapsidation of theretroviral RNAs occurs by virtue of a psi sequence located at the 5′ endof the viral genome.

The LTRs themselves are identical sequences that can be divided intothree elements, which are called U3, R and U5. U3 is derived from thesequence unique to the 3′ end of the RNA. R is derived from a sequencerepeated at both ends of the RNA and U5 is derived from the sequenceunique to the 5′ end of the RNA. The sizes of the three elements canvary considerably among different retroviruses.

For ease of understanding, a simple, generic diagram (not to scale) of aretroviral genome showing the elementary features of the LTRs, gag, poland env is presented in FIG. 5.

For the viral genome, the site of transcription initiation is at theboundary between U3 and R in the left hand side LTR (as shown above) andthe site of poly (A) addition (termination) is at the boundary between Rand U5 in the right hand side LTR (as shown above). U3 contains most ofthe transcriptional control elements of the provirus, which include thepromoter and multiple enhancer sequences responsive to cellular and insome cases, viral transcriptional activator proteins. Some retroviruseshave any one or more of the following genes that code for proteins thatare involved in the regulation of gene expression: tat, rev, tax andrex.

a p As shown in the diagram above, the basic molecular organisation of aretroviral RNA genome is (5′) R-U5-gag, pol, env-U3-R (3′). In aretroviral vector genome gag, pol and env are absent or not functional.The R regions at both ends of the RNA are repeated sequences. U5 and U3represent sequences unique, respectively, to the 5′ and 3′ ends of theRNA genome. These three sets of end sequences go to form the longterminal repeats (LTRs) in the proviral DNA, which is the form of thegenome which integrates into the genome of the infected cell. The LTRsin a wild type retrovirus consist of (5′) U3-R-U5 (3′), and thus U3 andU5 both contain sequences which are important for proviral integration.Other essential sequences required in the genome for proper functioninginclude a primer binding site for first strand reverse transcription,primer binding site for second strand reverse transcription and apackaging signal.

With regard to the structural genes gag, pol and env themselves and inslightly more detail, gag encodes the internal structural protein of thevirus. Gag is proteolytically processed into the mature proteins MA(matrix), CA (capsid), NC (nucleocapsid). The gene pol encodes thereverse transcriptase (RT), which contains both DNA polymerase, andassociated RNase H activities and integrase (IN), which mediatesreplication of the genome. The gene env encodes the surface (SU)glycoprotein and the transmembrane (TM) protein of the virion, whichform a complex that interacts specifically with cellular receptorproteins. This interaction leads ultimately to fusion of the viralmembrane with the cell membrane.

The envelope protein is a viral protein which coats the viral particleand plays an essential role in permitting viral entry into a targetcell. The envelope glycoprotein complex of retroviruses includes twopolypeptides: an external, glycosylated hydrophilic polypeptide (SU) anda membrane-spanning protein (TM). Together, these form an oligomeric“knob” or “knobbed spike” on the surface of a virion. Both polypeptidesare encoded by the env gene and are synthesised in the form of apolyprotein precursor that is proteolytically cleaved during itstransport to the cell surface. Although uncleaved Env proteins are ableto bind to the receptor, the cleavage event itself is necessary toactivate the fusion potential of the protein, which is necessary forentry of the virus into the host cell. Typically, both SU and TMproteins are glycosylated at multiple sites. However, in some viruses,exemplified by MLV, TM is not glycosylated.

Although the SU and TM proteins are not always required for the assemblyof enveloped virion particles as such, they do play an essential role inthe entry process. In this regard, the SU domain binds to a receptormolecule—often a specific receptor molecule—on the target cell. It isbelieved that this binding event activates the membrane fusion-inducingpotential of the TM protein after which the viral and cell membranesfuse. In some viruses, notably MLV, a cleavage event—resulting in theremoval of a short portion of the cytoplasmic tail of TM—is thought toplay a key role in uncovering the full fusion activity of the protein(Brody et at 1994 J. Virol. 68: 4620-4627, Rein et at 1994 J. Virol. 68:1773-1781). This cytoplasmic“tail”, distal to the membrane-spanningsegment of TM remains on the internal side of the viral membrane and itvaries considerably in length in different retroviruses.

Thus, the specificity of the SU/receptor interaction can define the hostrange and tissue tropism of a retrovirus. In some cases, thisspecificity may restrict the transduction potential of a recombinantretroviral vector. Here, transduction includes a process of using aviral vector to deliver a non-viral gene to a target cell. For thisreason, many gene therapy experiments have used MLV. A particular MLVthat has an envelope protein called 4070A is known as an amphotropicvirus, and this can also infect human cells because its envelope protein“docks” with a phosphate transport protein that is conserved between manand mouse. This transporter is ubiquitous and so these viruses arecapable of infecting many cell types. In some cases however, it may bebeneficial, especially from a safety point of view, to specificallytarget restricted cells. To this end, several groups have engineered amouse ecotropic retrovirus, which unlike its amphotropic relativenormally only infects mouse cells, to specifically infect particularhuman cells. Replacement of a fragment of an envelope protein with anerythropoietin segment produced a recombinant retrovirus which thenbound specifically to human cells that expressed the erythropoietinreceptor on their surface, such as red blood cell precursors (Maulik andPatel 1997 “Molecular Biotechnology: Therapeutic Applications andStrategies” 1997. Wiley-Liss Inc. pp 45.).

In addition to gag, pol and env, the complex retroviruses also contain“accessory” genes which code for accessory or auxillary proteins.Accessory or auxillary proteins are defined as those proteins encoded bythe accessory genes in addition to those encoded by the usualreplicative or structural genes, gag, pol and env. These accessoryproteins are distinct from those involved in the regulation of geneexpression, like those encoded by tat, rev, tax and rex. Examples ofaccessory genes include one or more of vif, vpr, vpx, vpu and nef. Theseaccessory genes can be found in, for example, HIV (see, for examplepages 802 and 803 of “Retroviruses” Ed. Coffin et at Pub. CSHL 1997).Non-essential accessory proteins may function in specialised cell types,providing functions that are at least in part duplicative of a functionprovided by a cellular protein. Typically, the accessory genes arelocated between pol and env, just downstream from env including the U3region of the LTR or overlapping portions of the env and each other.

The complex retroviruses have evolved regulatory mechanisms that employvirally encoded transcriptional activators as well as cellulartranscriptional factors. These trans-acting viral proteins serve asactivators of RNA transcription directed by the LTRs. Thetranscriptional trans-activators of the lentiviruses are encoded by theviral tat genes. Tat binds to a stable, stem-loop, RNA secondarystructure, referred to as TAR, one function of which is to apparentlyoptimally position Tat to trans-activate transcription.

As mentioned earlier, retroviruses have been proposed as a deliverysystem (other wise expressed as a delivery vehicle or delivery vector)for inter alia the transfer of a NOI, or a plurality of NOIs, to one ormore sites of interest. The transfer can occur in vitro, ex vivo, invivo, or combinations thereof. When used in this fashion, theretroviruses are typically called retroviral vectors or recombinantretroviral vectors. Retroviral vectors have even been exploited to studyvarious aspects of the retrovirus life cycle, including receptor usage,reverse transcription and RNA packaging (reviewed by Miller, 1992 CurrTop Microbiol Immunol 158: 1-24).

In a typical recombinant retroviral vector for use in gene therapy, atleast part of one or more of the gag, pol and env protein coding regionsmay be removed from the virus. This makes the retroviral vectorreplication-defective. The removed portions may even be replaced by aNOI in order to generate a virus capable of integrating its genome intoa host genome but wherein the modified viral genome is unable topropagate itself due to a lack of structural proteins. When integratedin the host genome, expression of the NOI occurs—resulting in, forexample, a therapeutic effect. Thus, the transfer of a NOI into a siteof interest is typically achieved by: integrating the NOI into therecombinant viral vector; packaging the modified viral vector into avirion coat; and allowing transduction of a site of interest—such as atargeted cell or a targeted cell population.

It is possible to propagate and isolate quantities of retroviral vectors(e.g. to prepare suitable titres of the retroviral vector) forsubsequent transduction of, for example, a site of interest by using acombination of a packaging or helper cell line and a recombinant vector.

In some instances, propagation and isolation may entail isolation of theretroviral gag, pol and env genes and their separate introduction into ahost cell to produce a “packaging cell line”. The packaging cell lineproduces the proteins required for packaging retroviral DNA but itcannot bring about encapsidation due to the lack of a psi region.However, when a recombinant vector carrying a NOI and a psi region isintroduced into the packaging cell line, the helper proteins can packagethe psi-positive recombinant vector to produce the recombinant virusstock. This can be used to infect cells to introduce the NOI into thegenome of the cells. The recombinant virus whose genome lacks all genesrequired to make viral proteins can infect only once and cannotpropagate. Hence, the NOI is introduced into the host cell genomewithout the generation of potentially harmful retrovirus. A summary ofthe available packaging lines is presented in “Retroviruses” (1997 ColdSpring Harbour Laboratory Press Eds: J M Coffin, S M Hughes, H E Varmuspp 449). However, this technique can be problematic in the sense thatthe titre levels are not always at a satisfactory level. Nevertheless,the design of retroviral packaging cell lines has evolved to address theproblem of inter alia the spontaneous production of helper virus thatwas frequently encountered with early designs. As recombination isgreatly facilitated by homology, reducing or eliminating homologybetween the genomes of the vector and the helper has reduced the problemof helper virus production.

More recently, packaging cells have been developed in which the gag, poland env viral coding regions are carried on separate expression plasmidsthat are independently transfected into a packaging cell line so thatthree recombinant events are required for wild type viral production.This strategy is sometimes referred to as the three plasmid transfectionmethod (Soneoka et at 1995 Nucl. Acids Res. 23: 628-633).

Transient transfection can also be used to measure vector productionwhen vectors are being developed. In this regard, transient transfectionavoids the longer time required to generate stable vector-producing celllines and is used if the vector or retroviral packaging components aretoxic to cells. Components typically used to generate retroviral vectorsinclude a plasmid encoding the Gag/Pol proteins, a plasmid encoding theEnv protein and a plasmid containing a NOI. Vector production involvestransient transfection of one or more of these components into cellscontaining the other required components. If the vector encodes toxicgenes or genes that interfere with the replication of the host cell,such as inhibitors of the cell cycle or genes that induce apotosis, itmay be difficult to generate stable vector-producing cell lines, buttransient transfection can be used to produce the vector before thecells die. Also, cell lines have been developed using transientinfection that produce vector titre levels that are comparable to thelevels obtained from stable vector-producing cell lines (Pear et at1993, PNAS 90: 8392-8396).

In view of the toxicity of some HIV proteins—which can make it difficultto generate stable HIV-based packaging cells—HIV vectors are usuallymade by transient transfection of vector and helper virus. Some workershave even replaced the HIV Env protein with that of vesicular stomatisvirus (VSV). Insertion of the Env protein of VSV facilitates vectorconcentration as HIV/VSV-G vectors with titres of 5×10⁵ (10⁸ afterconcentration) were generated by transient transfection (Naldini et at1996 Science 272: 263-267). Thus, transient transfection of HIV vectorsmay provide a useful strategy for the generation of high titre vectors(Yee et al 1994 PNAS. 91: 9564-9568).

If the retroviral component includes an env nucleotide sequence, thenall or part of that sequence can be optionally replaced with all or partof another env nucleotide sequence. Replacement of the env gene with aheterologous env gene is an example of a technique or strategy calledpseudotyping. Pseudotyping is not a new phenomenon and examples may befound in WO-A-98/05759, WO-A-98/05754, WO-A-97/17457, WO-A-96/09400,WO-A-91/00047 and Mebatsion et at 1997 Cell 90,841-847.

Pseudotyping can confer one or more advantages. For example, with thelentiviral vectors, the env gene product of the HIV based vectors wouldrestrict these vectors to infecting only cells that express a proteincalled CD4. But if the env gene in these vectors has been substitutedwith env sequences from other RNA viruses, then they may have a broaderinfectious spectrum (Verma and Somia 1997 Nature 389: 239-242). By wayof example—workers have pseudotyped an HIV based vector with theglycoprotein from VSV (Verma and Somia 1997 ibid). Alternatively, envcan be modified so as to affect (such as to alter) its specificity.

Thus, the term “recombinant retroviral vector” describes an entity (suchas a DNA molecule) which contains sufficient retroviral sequences toallow an RNA transcript of the vector to be packaged in the presence ofessential retroviral proteins into a retroviral particle capable ofinfecting a target cell. Infection of the target cell includes reversetranscription and integration into the target cell genome.

The term “recombinant retroviral vector” also covers a retroviralparticle containing an RNA genome encoded by the DNA molecule. Theretroviral vector will also contain non-viral genes which are to bedelivered by the vector to the target cell. A recombinant retroviralvector is incapable of independent replication to produce infectiousretroviral particles. Usually, a recombinant retroviral vector lacksfunctional gag-pol and/or env genes, or other genes encoding proteinsessential for replication.

The term “targeted retroviral vector” means a recombinant retroviralvector whose ability to infect a cell or to be expressed in the targetcell is restricted to certain cell types within the host organism. Anexample of targeted retroviral vectors is one with a geneticallymodified envelope protein which binds to cell surface molecules foundonly on a limited number of cell types in the host organism. Anotherexample of a targeted retroviral vector is one which contains promoterand/or enhancer elements which permit expression of one or moreretroviral transcripts in only a proportion of the cell types of thehost organism.

Thus, the present invention provides a useful delivery system. Thedelivery system is capable of targeting an NOI and/or a POI to atumour—i.e. capable of homing a NOI and/or POI in on a tumour.

The vector may be used to administered directly to an entity—such as anorganism or a cell thereof—such as ex vivo or in vivo. In this sense thevector may be delivered directly, for example, to a tumour site.Alternatively, the vector may be administered to an entity by way of acarrier—such as ex vivo or in vivo. An example of a carrier would be aliposome or a cell in which would be contained the vector. An example ofa suitable carrier cell would be a haematpoietic cell, such as a myeloidcell. These carrier cells may increase the further specificity of thevector of the present invention.

These and other aspects of the present invention will now be elaboratedon further.

The perceived potential of monoclonal antibody-based therapies fortreatment of neoplastic disease has not been fully realised (reviewed inScheinberg and Chapman 1995, In Monoclonal antibodies (ed. Birch andLennox) Chapter 2.1; George et al., 1994 Immunol. Today 15; 559-561).Consequently, monoclonal antibodies have been conjugated toradioisotopes, cytotoxic drugs or toxins in an attempt to improveefficacy. However, clinical trials with such conjugates have generallyled to disappointing results. One of the principal reasons for the lackof efficacy of antibodies and antibody conjugates in the treatment ofsolid tumours is the poor penetration of solid tumours byimmunoglobulins and other proteins such as immunotoxins of highmolecular weight (e.g. Juweid et al. 1992, Cancer Res. 52; 5144-5153;Epenetos et al. 1986 Cancer Res. 46; 3183-3191). Other reasons for lackof efficacy include the non-specific toxicity, immunogenicity andinappropriate pharmacokinetics of many immunotoxins andantibody-radionuclide conjugates introduced into the systemiccirculation (reviewed in Scheinberg and Chapman 1995. In Monoclonalantibodies (ed. Birch and Lennox) Chapter 2.1).

In contrast to the general lack of in vivo efficacy, many monoclonalantibodies show pronounced ability to inhibit the growth of tumour cellsin certain in vitro assays (reviewed in Sandlie and Michaelsen 1996 InAntibody engineering: a practical approach. Ed McCafferty et at Chapter9). It is well established that binding of specific antigen by anantibody can lead to activation of a variety of effector functionsmediated via the Fc portion of the antibody heavy chain. The Fc regionsof different immunoglobulin classes mediate different effectorfunctions, including activation of complement cascades and binding to Fcreceptors on various immune effector cells (Duncan et at 1988 Nature332; 563 and 738). In in vitro assays, engagement of Fc receptorspresent on immune effector cells by antibody bound to tumour targetcells can lead to destruction of the target cell by a variety ofmechanisms collectively termed antibody dependent cellular cytotoxicity(ADCC). For example engagement of Fc-receptors for IgG, on humanmonocytes and macrophages, neutrophils and natural killer (NK) cells byantibodies of the IgG1 and IgG3 and to a much lesser extent IgG2 andIgG4 sub-classes, stimulates ADCC (Munn et at 1991 Cancer Res. 51;1117-1123; Primus et al., 1993 Cancer Res. 53; 3355-3361). However, therelatively poor ability of such antibodies to destroy tumours in vivosuggests that ADCC does not play a significant role in many of thecurrent antibody-based therapies (George et al, 1994 Immunol. Today 15;559-561). There are several possible reasons for this, including thepoor penetration of antibodies into solid tumours (Yuan et al. 1995Cancer Res. 55; 3752-3756) and the fact that the majority of thehigh-affinity receptor (FcgRI) molecules present on macrophages arenormally occupied by serum IgG which will be poorly competed by specificantibody (Munn et al 1991 Cancer Res. 51; 1117-1123).

It has previously been shown that tumour cells transduced with genesencoding monoclonal antibodies can participate in ADCC reactionsmediated by xenogeneic NK cells in vitro (Primus et al. 1993 Cancer Res.53: 3355-3361). However, NK cells play little role in the destruction oftumour cells in vivo, in part because their killing functions areinhibited by the presence of self MHC Class I on autologous tumour cells(Correa and Raulet 1995 Immunity 2; 61-71).

It has also been postulated that tumour infiltrating lymphocytes (TILs)could be used as a vehicle to deliver antibody genes to a tumour tosecrete anti-tumour antibodies at the tumour site (Tsang et at 1993 J.Immunother. 13; 143-152.) However, ex vivo transduction of TILs followedby autologous transplantation using marker genes has shown that isolatedTILs show no specific homing mechanism which could allow them to returnto tumour deposits (Economou et at 1996 J. Clin. Invest. 97; 515-521)and so any such approach is of limited value. The present invention isin contrast to these findings since there is provided a vector that cantarget or deliver an NOI and/or a POI to a tumour mass (or site).

Transduction of a gene encoding a single-chain immunotoxin into humanlymphokine-activated T-cells (LAK cells) has also been reported (Chen etat 1997 Nature 385,78-80). In addition to the problems of re-introducingthe LAK cells to the site of the tumour, such an approach also suffersfrom the potential drawbacks associated with being restricted to ex vivouse. These include the necessity of culturing the T-cells in high levelsof a cytokine such as IL-2 to generate LAK cells with consequentproblems in generating sufficient cells for therapy.

In one aspect, the present invention relates to the use of geneticvectors to deliver genes (such as therapeutic genes) encoding secretedtumour binding proteins (TBPs) to the interior of a tumour mass andidentifies ways to target expression of TBPs to the interior of thetumour. Expression of the gene or genes encoding the TBP within thetumour mass then leads to local production of TBP with consequentreduction of tumor growth, survival or dissemination by a variety ofmechanisms. Because the TBP is secreted, TBP produced by transducedcells can act not only on the transduced cell but on neighbouring tumourcells as well and hence achieve a bystander effect.

There are a number of cell types present within a tumor mass in additionto the cancerous cells. These can include cells of the tumourvasculature (e.g. endothelial cells) and immune cells which infiltratethe tumour, such as tumour-infiltrating lymphocytes (TIL) andmacrophages (Normann 1985 Cancer Metastasis Re. 4: 277-291; Leek et at1996 Cancer Res. 56: 4625-4629). Any of these cell types can be targetedfor expression of the TBP and can serve as a local factory within thetumour for production of TBP. Preferably, the cells in the tumour masswhich are used to produce the TBP are the cancerous cells, endothelialcells or macrophages. Alternatively, the progenitors of monocytes orendothelial cells may be targeted, such as CD34-positive peripheralblood mononuclear cells (Asahara et al. 1997 Science 275: 964-967).

Preferably, the TBP comprises one or more binding domains capable ofbinding to one or more TACSMs which are present on the cancerous cells.Thus the TBP, produced from one or more of the cell types within thetumour mass is secreted and is directed to the cancerous cells by itsaffinity for the TACSM. The TACSM may be selectively present on arestricted number of cell types. Thus the amount of TACSM present on themajority of the cancerous cells within the tumour mass is higher than onsurrounding tissues. Preferably, the TACSM is detectably present only ontumour cells and a limited number of other tissue types in theindividual containing the tumour. More preferably, the TACSM isessentially tumour-specific in the individual containing the tumour.

The one or more binding domains of the TBP may consist of, for example,a natural ligand for a TACSM, which natural ligand may be an adhesionmolecule or a growth-factor receptor ligand (e.g. epidermal growthfactor), or a fragment of a natural ligand which retains bindingaffinity for the TACSM. Alternatively, the binding domains may bederived from heavy and light chain sequences from an immunoglobulin (Ig)variable region. Such a variable region may be derived from a naturalhuman antibody or an antibody from another species such as a rodentantibody. Alternatively the variable region may be derived from anengineered antibody such as a humanised antibody or from a phage displaylibrary from an immunised or a non-immunised animal or a mutagenisedphage-display library. As a second alternative, the variable region maybe derived from a single-chain variable fragment (scFv). The TBP maycontain other sequences to achieve multimerisation or to act as spacersbetween the binding domains or which result from the insertion ofrestriction sites in the genes encoding the TBP, including Ig hingesequences or novel spacers and engineered linker sequences.

The TBP may comprise, in addition to one or more immunoglobulin variableregions, all or part of an Ig heavy chain constant region and so maycomprise a natural whole Ig, an engineered Ig, an engineered Ig-likemolecule, a single-chain Ig or a single-chain Ig-like molecule.Alternatively, or in addition, the TBP may contain one or more domainsfrom another protein such as a toxin.

In one aspect of the invention, there is provided a gene delivery systemfor targeting one or more genes encoding a TBP to a tumour, comprising agenetic vector encoding a TBP and an in vivo gene-delivery system. Thegene delivery system may be a non-viral gene delivery system such as DNAcompacted with a DNA-compaction agent, or a liposome or immunoliposomewhich may contain DNA compacted with a DNA-compaction agent (such as apoly-lysine). The vector may be a plasmid DNA vector. Alternatively thevector may be a recombinant viral vector such as an adenovirus vector,an adeno-associated virus (AAV) vector, a herpes-virus vector or aretroviral vector in which case gene delivery is mediated by viralinfection of a target cell. Preferably the vector is a recombinantretroviral vector, which may be a targeted retroviral vector.Preferably, the retroviral vector is resistant to human complement, forexample by production in a human cell line.

Typically, the vector will contain a promoter to direct expression ofthe or each gene (such as a therapeutic gene) and may contain additionalgenetic elements for the efficient or regulated expression of TBP genes,including enhancers, translation initiation signals, internal ribosomeentry sites (IRES), splicing and polyadenylation signals. The promoterand/or enhancer may be tissue-restricted in its activity. For example atumour-specific promoter-enhancer, such as a 5T4 antigen genepromoter-enhancer or the CEA-gene promoter-enhancer may be used.Alternatively, or additionally, an element or elements for regulatedexpression may be present, such as a hypoxia regulated enhancer. Anexample of a hypoxia regulated expression element (HRE) is a bindingelement for the transcription factor HIF1. The enhancer elements orelements conferring regulated expression may be present in multiplecopies. Preferably, expression of the or a gene (such as a therapeuticgene) is inducible by hypoxia (or low oxygen supply) such as may befound in a tumour mass. Most preferably, the promoter and/or enhancerdirecting expression of the gene (such as a therapeutic gene) containsboth hypoxia-responsive elements and elements which give higherexpression in tumour cells than in neighbouring non-tumour cells.

Additional vector components will be provided for other aspects ofvector function such as vector maintenance, nuclear localisation,replication, and integration as appropriate using components which arewell known in the art.

In a preferred embodiment of this aspect of the invention, a retroviralvector is provided for in vivo delivery of the gene or genes encodingthe TBP to the tumour. Suitable retroviral vectors are known in the art(see for example Gunzberg and Salmons 1996 In Gene Therapy ed. Lemoineand Cooper. Bios; and Cosset et al. 1995 J. Virol. 69; 7430-7436). In aparticularly preferred embodiment, expression of the TBP may be enhancedin the hypoxic regions of the tumour by the inclusion of hypoxiaregulated genetic elements in the retroviral vector. In this case, thehypoxia-regulated elements may be inserted into one or both of theretroviral LTRs in place of the LTR enhancer or in another position inthe vector, by standard molecular biology techniques. The gene or genesencoding the TBP may be expressed from a promoter-enhancer which leadsto enhanced expression in the tumour cells compared with neighbouringnon-tumour cells or is preferably essentially tumour-specific. Examplesof suitable promoters include the promoter-enhancer of the gene for 5T4antigen, the promoter-enhancer of the MUC1 gene or the CEA gene.

In an other aspect of the invention there is provided a method oftreating cancer comprising administering the TBP gene or genes in a genedelivery system of the first aspect of the invention either systemicallyor directly to the site of a tumour.

In an other aspect of the invention, is provided a gene delivery systemfor introducing one or more genes encoding a TBP into cells of thehaematopoietic (preferably myeloid haematopoietic) cell lineage eitherin vivo or ex vivo. Preferably the haematopoietic (preferably myeloidhaematopoietic) cells are of the monocyte-macrophage lineage or aprecursor of such cells such as a CD34-positive stem cell. For ex vivodelivery, the genes can be inserted into a plasmid vector and deliveredby one of a variety of DNA transfection methods includingelectroporation, DNA biolistics, lipid-mediated transfection orcompacted DNA-mediated transfection. Alternatively a viral vector can beused to transduce haematopoietic (preferably myeloid haematopoietic)cells or CD34-positive stem cells ex vivo, such as an adenovirus vector,a retroviral vector or a lentiviral vector. The vector will contain apromoter to direct expression of the or each gene (such as a therapeuticgene) and may contain additional genetic elements for efficient orregulated expression including enhancers, translation initiationsignals, internal ribosome entry sites (IRES), splicing andpolyadenylation signals. The promoter, or an enhancer or splicingsignals may be tissue-restricted and preferentially active inmononuclear phagocytes such as macrophages. The promoter and/or enhancermay contain elements for regulated expression such as ahypoxia-regulated enhancer. An example of a hypoxia regulated expressionelement is HIF1 transcription factor response element. Such an elementmay be present in multiple copies. Examples of hypoxia-regulatedpromoters and enhancers include those from the enolase gene, theerythropoietin gene, and genes encoding glycolytic enzymes (Semenza etal., 1994 J. Biol. Chem 269; 23757-23763) such as the PGK gene. IsolatedHREs can be multimerised in order to increase the response to hypoxia.Additional vector components may be provided for other aspects of vectorfunction such as vector maintenance, nuclear localisation, replicationand integration as appropriate using components which are well known inthe art.

After introduction of the vector into the cells ex vivo, the cells canbe re-introduced into the patient directly or they can be stimulated todifferentiate along the monocyte-macrophage differentiation pathwayusing appropriate combinations of cytokines and growth factors prior tore-introduction into the patient. CD34-positive cells are stimulated todifferentiate using cytokines including IL-3, GMCSF and MCSF. Monocytesare differentiated either by culture attached to plastic or using GMCSFeither alone or in combination with other cytokines including MCSF.

For introduction of genes (such as therapeutic genes) intohaematopoietic (preferably myeloid haematopoietic) cells orCD34-positive stem cells in vivo, a suitable in vivo delivery system canbe used to deliver the transcription units described above. The genedelivery system may be a non-viral gene delivery system such as DNAcompacted with a DNA-compaction agent, or a liposome or immunoliposomewhich may contain DNA compacted with a DNA-compaction agent.Alternatively the vector may be a recombinant viral vector such as atargeted adenovirus vector, an adeno-associated viral (AAV) vector, aherpes-virus vector or a retroviral vector such as a lentiviral vector.Preferably the vector is a targeted recombinant retroviral vector, whichis preferably resistant to human complement, for example by preparationof the vector from a human packaging cell line.

CD34-positive stem cells can also differentiate to form endothelialcells (Ashara et al. 1997 Science 275; 964-967). Such a route ofdifferentiation for CD34 positive stem cells containing TBP encodinggenes according to the invention is envisaged in addition todifferentiation to form monocytes and macrophages.

Additional vector components may be provided for other aspects of vectorfunction such as vector maintenance, nuclear localisation, replication,and integration as appropriate using components which are well known inthe art.

In a preferred embodiment of this aspect of the invention, a plasmidvector or a retroviral vector carrying a gene encoding a TBP under thecontrol of a hypoxia regulated promoter or a promoter preferentiallyactive in macrophages is introduced into autologous peripheral bloodmonocytes. The transfected monocytes are re-introduced into the patientwhere they migrate to the hypoxic regions of tumours permitting enhancedproduction of the TBP in the interior of the tumour mass. Themacrophages are optionally treated with cytokines prior to re-injectioninto the patient. Alternatively or additionally the vector may includeDNA sequences capable of expressing a cytokine gene such as a gene forIFNg, CSF-1 or GM-CSF in order to elicit the differentiation of thetransfected cells. The cytokine gene may also be regulated by geneticelements which show enhanced activity at the site of the tumour.

In an other aspect of the invention, there is provided a method fortreating cancer in a human or non-human mammal, comprising withdrawingan amount of blood from an individual suffering from cancer, preparingfrom the blood a cell preparation enriched in monocytes and macrophagesor their stem-cell progenitors, introducing TBP genes into the cellpreparation using a gene delivery system of the third aspect of theinvention so as to bring about transfection or transduction of themonocytes and macrophages, or their stem-cell progenitors with the TBPgenes, and re-introducing the transfected or transduced cells eithersystemically or directly to the site of the tumour. The cellpreparations may optionally be treated with cytokines prior toreintroduction in order to elicit differentiation towards activemacrophages.

In an other aspect of the invention is provided a method for treatingcancer in a mammal, comprising administering to an individual a genedelivery system of the invention capable of expressing a TBP in cellsderived from a haematopoietic (preferably myeloid haematopoietic)origin.

In a further aspect of the invention there is provided a genetic vectorcomprising a gene (such a therapeutic gene) or genes encoding a TBP,operably linked to an expression regulatory element selectivelyfunctional in a cell type present within a tumour mass. The TBP in thisaspect of the invention inhibits tumour function by binding to a TACSMhaving an essential role in tumour cell survival or dissemination. TheTACSM in this aspect of the invention may be a cell surface moleculewhich has a role in tumour cell growth, migration or metastasis, and ispresent on cancerous cells or on another cell type within the tumourmass. Preferably the TACSM is present on cancerous cells or tumourvasculature or on macrophages and is a molecule such as a growth-factorreceptor, a plasminogen activator, a metalloproteinase or the 5T4antigen. The gene or genes encoding the TBP may be delivered to theinterior of the tumour by any of the routes described in the above twoaspects of the invention. Binding of the TBP to the corresponding TACSMblocks the function of the TACSM and thereby leads to inhibition ofgrowth, migration or metastasis of the tumour.

In a yet further aspect of the invention, a genetic vector comprising agene (such as a therapeutic gene or genes) is delivered to the interiorof the tumour wherein the gene (such as a therapeutic gene) encodes aTBP, which additionally contains one or more effector domains. Theeffector domain or domains may be activated on binding of the TBP to aTACSM leading to inhibition of tumour cell proliferation, survival ordissemination. The TACSM in this aspect of the invention is a cellsurface molecule for which a specific TBP is available such as a tumourspecific carbohydrate moiety, an oncofoetal antigen, a mucin, agrowth-factor receptor or another glycoprotein. The TACSM is preferablyan antigen restricted in its tissue distribution and found predominantlyon the tumour cells and on the majority of cells within the tumour.Alternatively, the TACSM is present on tumour macrophages or the tumourvasculature. In some instances, the TACSM is not shed from the cellsurface into the circulation to an appreciable extent. However, sheddingmay occur. By way of example, shedding of the 5T4 antigen into thestroma can serve to further localise the NOI and/or the POI to thetumour environment. The effector domain of the present invention maypossess enzymatic activity and may be for example a pro-drug activatingenzyme, or it may be a non-enzyme domain. Examples of TBPs containingeffector domains with enzyme activity include antibody-enzyme conjugatesor fusions. Antibody-enzyme conjugates have been described includingconjugates with alkaline phosphatase (Senter et al., 1988 Proc. Natl.Acad. Sci. 85: 48424846); carboxypeptidase G2 (Bagshawe et al. 1988 Br.J. Cancer 58: 700703); P-lactamase (Shepherd et at 1991 Bioorg. Med.Chem. Left. 1: 21-26); and Penicillin-V-amidase (Kerr et al. 1990 CancerImmunol. Immunother. 31: 202-206. Antibody-enzyme fusions have also beendescribed (Goshorn et at 1993 Cancer Res 53: 2123-2127; Wels et al 1992Bio/Technology 10: 1 1 28-1132). Each of these examples can be used inthis aspect of the invention. Additional or alternative enzymes whichmay be included in TBPenzyme fusions include human Carboxypeptidase A1or a mutant thereof (Smith et at 1997 J. Biol. Chem. 272: 15804-15816);cytosine deaminase (Mullen et al. 1994 Cancer Res. 54: 1503- 1506); HSVthymidine kinase (Borrelli et al. 1988 Proc. Natl. Acad. Sci. 85:7572-7576.); nitroreductase; P450-Reductase and a P450.

Preferably the pro-drug activating enzyme domain or domains aregenetically fused to the C-terminus of an immunoglobulin orimmunoglobulin domain such as a scfv or a single-chain antibody orFab-fragment. In a particularly preferred embodiment of this aspect ofthe invention, the immunoglobulin domain or domains are human orhumanised and the enzyme is a human enzymp—such as a Carboxypeptidase aP450 or P450-Reductase. The enzyme may be a mutant enzyme which convertsa pro-drug more efficiently than does the native human enzyme. Inaccordance with the present invention, any enzyme that has utility in anADEPT strategy can be used.

In each case, a suitable pro-drug is used in the treatment of thepatient in combination with the appropriate pro-drug activating enzyme.Examples of pro-drugs include etoposide phosphate (used with alkalinephosphatase Senter et al., 1988 Proc. Nat. Acad. Sci. 85: 4842-4846);5-fluorocytosine (with Cytosine deaminase Mullen et al. 1994 Cancer Res.54: 1503-1506); Doxorubicin-N-p-hydroxyphenoxyacetamide (withPenicillin-V-Amidase (Kerr et al. 1990 Cancer Immunol. lmmunother. 31:202-206); Para-N-bis (2chloroethyl) aminobenzoyl glutamate (withCarboxypeptidase G2); Cephalosporin nitrogen mustard carbamates (withP-lactamase); SR4233 (with P450 Reducase); Ganciclovir (with HSVthymidine kinase, Borrelli et al. 1988 Proc. Natl. Acad. Sci. 85:7572-7576) mustard pro- drugs with nitroreductase (Friedlos et al. 1997J Med Chem 40: 1270-1275) and Cyclophosphamide (with P450 Chen et al.1996 Cancer Res 56: 1331-1340).

Alternatively the effector domain may be a non-enzyme domain. Examplesof non-enzyme effector domains include toxins such an exotoxin from apseudomonad bacterium, all or part of a cytokine such as IL-2 or IFNγ,or effector domains from immunoglobulin heavy chains.

In a preferred embodiment of this aspect of the invention, the TBPcontains an effector domain capable of activating macrophage FcgR I, IIor III receptors. On binding of the TBP to antigen on the tumour cells,macrophages present within the hypoxic regions of the tumour areactivated to destroy the tumour cells directly by phagocytosis or ADCCor are activated to secrete pro-inflammatory cytokines which serve toenhance the natural immunological response to the tumour. The TBP maycontain an Fc region from an immunoglobulin, a mutant Fc region, areceptor-binding fragment of the Fc region or may contain anotherFcR-binding domain.

Preferably the TBP contains an entity, preferably an effector domainentity, that confers protein stability ex vivo and/or in vivo.

In accordance with the present invention, the TBP may include an intactFc region from an IgG, (such as human IgG1 or IgG3), preferably from IgE(such as human IgGE), or a part thereof.

In one preferred embodiment of this aspect of the invention, the TBP isa Sab (single chain antibody) containing a human IgG1 constant regionand a binding domain which recognises the 5T4 antigen.

In a particularly preferred embodiment of this aspect of the invention,the TBP is a Sab (single chain antibody) containing a human IgE constantregion and a binding domain which recognises the 5T4 antigen.

The effector domain may be encoded by a portion of a cDNA fused in-frameto the DNA encoding the tumour-binding domain. Alternatively a genomicfragment containing introns may be used such as a human IgGI heavy chainconstant region genomic fragment.

Here the term “intron” is used in its normal sense—e.g. an interveningsequence of DNA within a gene which is removed by RNA splicing and so isnot present in the mature messenger RNA and does not code for protein.Introns can be conditional or alternatively spliced in different celltypes.

Introduction of TBP-encoding genes into monocytes or macrophages may becombined with further treatments to elicit macrophage differentiationand activation. For example, cells maintained ex vivo may be treatedwith cytokines such as IFNγ, CSF-1 or GM-CSF prior to re-introductioninto the patient. Alternatively, genes encoding these cytokines may beintroduced into the monocytes/macrophages in the same or a differentvector from the TBP genes in vivo or ex vivo. Consequently in a stillfurther aspect of the invention there is provided a method of treatingcancer in a mammal which comprises administering to an individual acombination of a cytokine or a cytokine-encoding gene and one or moreTBP genes according to any of the previous aspects of the invention.

In accordance with the invention, standard molecular biology techniquesmay be used which are within the level of skill in the art. Suchtechniques are fully described in the literature. See for example;Sambrook et al. (1989) Molecular Cloning; a laboratory manual; Hames andGlover (1985-1997) DNA Cloning: a practical approach, Volumes I-IV(second edition). Methods for the engineering of immunoglobulin genes inparticular are given in McCafferty et at (1996) Antibody engineering: apractical approach.

In a preferred aspect, the present invention relates to the delivery ofTBP-encoding genes to the site of a tumour. This has considerableadvantages for medical applications (such as therapeutic applications)in which TBPs are indicated since it circumvents a number of problemsassociated with delivery of proteins systemically in humans. In contrastto the problems associated with production and delivery of proteins, themethods of the invention allow the delivery of genes to the site of thetumour, thus circumventing a number of production problems. The TBPs arethereby produced in situ in the autologous human cells, which serve as alocal factory for the production of the gene- based medicament (such asa therapeutic). This has significant advantages in minimising systemictoxicity. The activity of the protein is maximal since the glycosylationof the protein shows a human pattern appropriate to the individual beingtreated.

The methods of the invention can be used in conjunction with directinjection into the site of the tumour or systemic delivery of, forexample targeted vectors or engineered haematopoietic (preferablymyeloid haematopoietic) cells or their progenitors. Systemic deliverymay be particularly advantageous in a number of indications,particularly in the treatment of disseminated disease. In these casesthe gene delivery system or engineered cells can be administeredintravenously by bolus injection or by infusion in a suitableformulation. A pharmaceutically acceptable formulation may include anisotonic saline solution, a buffered saline solution or a tissue-culturemedium. Additional formulatory agents may be included such aspreservative or stabilising agents.

Thus, the present invention also encompasses a pharmaceuticalcomposition for treating one or more individuals by gene therapy,wherein the composition comprises a therapeutically effective amount ofthe vector according to the present invention or the expressed productthereof. The pharmaceutical composition may be for human or animalusage. Typically, a physician will determine the actual dosage whichwill be most suitable for an individual subject and it will vary withthe age, weight and response of the particular patient.

The composition may optionally comprise a pharmaceutically acceptablecarrier, diluent, excipient or adjuvant. The choice of pharmaceuticalcarrier, excipient or diluent can be selected with regard to theintended route of administration and standard pharmaceutical practice.The pharmaceutical compositions may comprise as—or in addition to—thecarrier, excipient or diluent any suitable binder (s), lubricant (s),suspending agent (s), coating agent (s), solubilising agent (s), andother carrier agents that may aid or increase the viral entry into thetarget site (such as for example a lipid delivery system).

Where appropriate, the pharmaceutical compositions can be administeredby any one or more of: inhalation, in the form of a suppository orpessary, topically in the form of a lotion, solution, cream, ointment ordusting powder, by use of a skin patch, orally in the form of tabletscontaining excipients such as starch or lactose, or in capsules orovules either alone or in admixture with excipients, or in the form ofelixirs, solutions or suspensions containing flavouring or colouringagents, or they can be injected parenterally, for exampleintracavernosally, intravenously, intramuscularly or subcutaneously. Forparenteral administration, the compositions may be best used in the formof a sterile aqueous solution which may contain other substances, forexample enough salts or monosaccharides to make the solution isotonicwith blood. For buccal or sublingual administration the compositions maybe administered in the form of tablets or lozenges which can beformulated in a conventional manner.

Thus, a preferred aspect of the present invention relates to a vectorcomprising (a) a NS coding for a TIP and (b) an NOI which encodes a POI;wherein the TIP is capable of recognising a tumour, such that in use thevector is capable of delivering the NOI and/or the POI to the tumour.

In one exemplary embodiment of the present invention TIP is IgG or IgEor a part thereof.

In another exemplary embodiment of the present invention, TIP is EGF ora part thereof.

In another exemplary embodiment of the present invention, TIP recognisesa trophoblast cell surface antigen and at least one of the effectordomains is a secreted co-stimulatory molecule. Further backgroundteaching and details on this embodiment now follow.

The latter-mentioned aspect of the present invention relates to aprocess for the activation of lymphocytes and the use of activatedlymphocytes in the treatment of cancer. It also relates to fusionproteins for the activation of lymphocytes, to nucleic acids encodingthe fusion proteins and to vectors carrying the nucleic acids.Lymphocytes require at least two distinct signals in order to respond toantigens by activation of effector functions (Bretscher and Cohn 1970Science 169: 1042-1049; Crabtree 1989 Science 243: 355-361). The primarysignal is specific for antigen. For B-lymphocytes, the B-cell antigenreceptor (surface immunoglobulin) recognises three-dimensional epitopeson a variety of macromolecules. For T-lymphocytes, the T-cell receptor(TCR) recognises peptide antigens displayed on the surface ofantigen-presenting cells by proteins of the major histocompatability(MHC) family (Weiss et al. 1986 Ann Rev. Immunol. 4: 593-619).

Stimulation of the primary signal in isolation normally leads toapoptosis (programmed cell death) of the lymphocyte or leads to theestablishment of a state of sustained unresponsiveness or anergy (Weisset al. supra). In order to achieve activation of the lymphocyte,accessory signals are required which may be delivered by cytokines or bycell-surface co-stimulatory ligands present on antigen-presenting cells(APC).

There are a number of such co-stimulatory molecules now identifiedincluding adhesion molecules, LFA-3, ICAM-1, ICAM-2. Majorco-stimulatory molecules present on APC are the members of the B7 familyincluding B7-1 (CD80), B7-2 (CD86) and B7-3. These molecules are ligandsof co-stimulatory receptors on lymphocytes including CD28 (WO92/00092),probably the most significant co-stimulatory receptor for restingT-cells. Different members of the B7 family of glycoproteins may deliversubtly different signals to T-cells (Nunes et al. 1996 J. Biol. Chem.271: 1591-1598).

Established tumours, despite the fact that they commonly express unusualantigens on their surfaces, are poorly immunogenic. It has beenpostulated previously that one method for stimulating immune recognitionof tumour cells would be to enhance antigen presentation andco-stimulation of lymphocytes in the context of tumour antigens.Transfection of the genes encoding B7-1 and B7-2, alone or incombination with cytokines, have been shown to enhance the developmentof immunity to experimental tumours in animal models (e.g. Leong et al.1997 Int. J. Cancer 71: 476-482; Zitvogel et al. 1996 Eur. J. Immunol.26: 1335-1341; Cayeux et al. 1997 J. Immunol 158: 2834-2841). However,in translating these results into a practical treatment for humancancer, there are a number of significant problems to be overcome. Amajor problem in such studies is the need to deliver B7 genes in vivo toa large number of cells of the tumour to achieve efficacy. A secondproblem is that it is important to target expression of B7 to the tumourcells to avoid inappropriate immune cell activation directed againstother cell types.

This aspect of the present invention solves these specific problems bydelivering a gene encoding a secreted co-stimulatory molecule (“SCM”)with binding affinity for a tumour antigen. In this way, a relativelysmall number of transfected cells within the tumour act as a localfactory to produce the co-stimulatory molecule which is shed from theproducer cell and binds to other cells in the tumour. The aspect of thepresent invention has the additional advantage that tumour cells neednot be the target for transfection.

The SCM of the invention is a novel engineered fusion protein comprisinga signal peptide for secretion from mammalian cells, at least oneantigen-binding domain from an immunoglobulin or an immunoglobulin-likemolecule and at least one further domain which acts as a co-stimulatorysignal to a cell of the immune system. The use of combinations of SCMscontaining different co-stimulatory domains is also envisaged. The SCMsare produced by expression of SCM-encoding genes in the autologous cellsof the individual to be treated and hence any post-translationalmodifications added to the protein by the host cell are authentic andprovide fully functional protein and appropriate pharmacokinetics.

WO-A-92/00092 describes truncated forms of B7-1, derived by placing atranslation stop codon before the transmembrane domain, secreted frommammalian cells. In that particular case, a heterologous signal peptidefrom the Oncostatin M gene was used. WO-A92/00092 also describes fusionproteins which contain the extracellular domain of B7-1 fused to the Fcregion of an immunoglobulin. Such molecules can bind to CD28 on T-cellsand serve to stimulate T-cell proliferation. However such stimulationoccurs only to a moderate extent unless the B7 or B7-derivative isimmobilised on a solid surface.

Gerstmayer et al. (1997 J. Immol. 158: 4584-4590) describes a fusion ofB7-2 to an scFv specific for ErbB2 followed by a myc epitope tag andpolyhistidine tag which is secreted when expressed in the yeast Pichiapastoris. This molecule retained binding for antigen and co-stimulatedproliferation of T-cells prestimulated with PMA and IL-2. However,glycosylation of such a molecule is of the yeast type, which is likelyto lead to inappropriate pharmacokinetics in humans.

In accordance with the present invention, any suitable co-stimulalatorydomain(s) may be used. By way of example, co-stimulatory domains can bechosen from extracellular portions of the B7 family of cell-surfaceglycoproteins, including B7-1, B7-2 and B7-3 or other co-stimulatorycell surface glycoproteins such as but not limited to co-stimulatoryreceptor-ligand molecules including CD2/LFA-3, LFA-1/ICAM-1 and ICAM-3.Studies have demonstrated that T cell co-stimulation by monocytes isdependent on each of two receptor ligand pathways CD2/LFA-3 andLFA-1/ICAM-1 (Van Seventer et at 1991 Eur J Immunol 21: 1711-1718). Inaddition, it has been shown that ICAM-3, the third LFA-1counterreceptor, is a co-stimulatory molecule for resting and activatedT lymphocytes (Hernandez-Caselles et at 1993 Eur J Immunol 23:2799-2806).

Other possible co-stimulatory molecules may include a novel glycoproteinreceptor designated SLAM, has been identified which, when engaged,potentiates T-cell expansion in a CD28-independent manner and induces aThO/Th1 cytokine production profile (Cocks et at 1995 Nature 376:260-263).

CD6, a cell surface glycoprotein, has also been shown to function as aco-stimulatory and adhesion receptor on T cells. Four CD6 isoforms(CD6a, b, c, d) have been described (Kobarg et at 1997 Eur J Immunol 27:2971-2980). A role for the very late antigen (VLA-4) integrin in theactivation of human memory B cells has also been suggested (Silvy et at1997 Eur J Immunol 27: 2757-2764). Endothelial cells also provide uniqueco-stimulatory signals that affect the phenotype of activated CD4+ Tcells (Karmann et at 1996 Eur J Immunol 26: 610-617). A B3 protein,present on the surface of lipopolysaccharide-activated B cells, whichcan provide co-stimulation to resting T cells leading to a predominantrelease of interleukin (IL)-4 and IL-5 and negligible amounts of IL-2and interferon gamma has been described (Vinay et at 1995 J Biol Chem270: 23429-23436). The co-expression of a novel co-stimulatory T cellantigen (A6H) on T cells and tumour cells has suggested a possiblefunction related to common properties of these cells (Labuda et at 1995Int Immunol 7: 1425-1432).

In one preferred embodiment of the invention, the co-stimulatory domainis a portion of B7-1 or B7-2, more preferably the complete extracellularportion of B7-1 or B7-2.

The SCM is formed by expression of a novel gene encoding a fusionprotein containing the antigen-binding domain or domains and theco-stimulatory domain or domains. If the antigen-binding domain iscomprised of a heavy and a light chain, the co-stimulatory domain isfused to one or other of the immunoglobulin chains, preferably to theheavy chain. If the antigen-binding domain is a scFv, the co-stimulatorydomain is fused to the scFv. The domains can be placed in the order(N-terminus to C-terminus): antigen-binding domain followed byco-stimulatory domain; or co-stimulatory domain followed byantigen-binding domain. Preferably, the co-stimulatory domain is placedat the N-terminus followed by the antigen-binding domain. A signalpeptide is included at the N-terminus, and may be for example thenatural signal peptide of the co-stimulatory extracellular domain. Thedifferent domains may be separated by additional sequences, which mayresult from the inclusion of convenient restriction-enzyme cleavagesites in the novel gene to facilitate its construction, or serve as apeptide spacer between the domains, or serve as a flexible peptidelinker or provide another function. Preferably the domains are separatedby a flexible linker.

Two or more different genes encoding different SCMs may be used toachieve improved co-stimulation, or both co-stimulation of naïve T-cellsand induction of memory responses. For example a gene encoding an SCMcontaining the B7-1 extracellular domain may be administered with a geneencoding an SCM containing the B7-2 extracellular domain.

Thus in one aspect of the invention, there is provided one or moregenetic vectors capable of expressing in mammalian cells one or moresecreted co-stimulatory molecules, each secreted co-stimulatory moleculecomprising at least one antigen-binding domain and at least one domainfrom the extracellular portion of a cell-surface co-stimulatorymolecule. The co-stimulatory domain may be obtained from a moleculeexpressed on the surface of an antigen-presenting cell such as a B7family member. Preferably the co-stimulatory domain is from B7-1, B7-2or B7-3. Most preferably it is comprised of B7-1 amino acid residues 1to approximately 215 of the mature B7-1 molecule (described inWO-A-96/00092) or amino acids 1 to approximately 225 of the maturecell-surface form of B7-2 (described in Gerstmeyer et al. 1997 J.Immunol. 158: 4584-4590).

The genetic vector according to this aspect of the invention comprisesat least a promoter and enhancer for expression in mammalian cells and apolyadenylation site. Suitable promoters and enhancers include the MIEpromoter-enhancer from human cytomegalovirus or promoters which areexpressed preferentially in cells present within the tumour. Suchpromoter-enhancers include those from the MUC1 gene, the CEA gene or the5T4 antigen gene. If two or more SCMs are expressed, the coding regionsfor these may be inserted into two separate vectors or a single vectormay be used to express the two or more genes. In the latter case eachgene is provided with a separate copy of the promoter, or an internalribosome entry site (IRES) is used to separate the two coding sequences.

The present invention also covers the use of mutants, variants,homologues or fragments of the sequences disclosed herein.

The terms “variant”, “homologue” or “fragment” in relation to thenucleotide sequences include any substitution of, variation of,modification of, replacement of, deletion of or addition of one (ormore) nucleic acid from or to the sequence providing the resultantnucleotide sequence codes for or is capable of coding for an entityhaving the same function as that presented herein, preferably being atleast as biologically active as the same. In particular, theterm“homologue”covers homology with respect to structure and/or functionproviding the resultant nucleotide sequence codes for or is capable ofcoding for an entity having the same function as that presented herein.With respect to sequence homology, preferably there is at least 75%,more preferably at least 85%, more preferably at least 90% homology tothe sequences shown herein. More preferably there is at least 95%, morepreferably at least 98%, homology to the sequences shown herein.

In particular, the term “homology” as used herein may be equated withthe term “identity”. Relative sequence homology (i. e. sequenceidentity) can be determined by commercially available computer programsthat can calculate % homology between two or more sequences. A typicalexample of such a computer program is CLUSTAL.

The terms “variant”, “homologue” or “fragment” are synonymous withallelic variations of the sequences.

The term “variant” also encompasses sequences that are complementary tosequences that are capable of hybridising to the nucleotide sequencespresented herein. Preferably, the term “variant”encompasses sequencesthat are complementary to sequences that are capable of hybridisingunder stringent conditions (e.g. 65° C. and 0.1×SSC {1×SSC=0.15 M NaCl,0.015 Na₃ citrate pH 7.0}) to the nucleotide sequence presented herein.

The present invention also covers nucleotide sequences that canhybridise to the nucleotide sequences of the present invention(including complementary sequences of those presented herein). In apreferred aspect, the present invention covers nucleotide sequences thatcan hybridise to the nucleotide sequence of the present invention understringent conditions (e.g. 65° C. and 0.1×SSC) to the nucleotidesequence presented herein (including complementary sequences of thosepresented herein).

The terms “variant”, “homologue” or “fragment” in relation to the aminoacid sequences include any substitution of, variation of, modificationof, replacement of, deletion of or addition of one (or more) amino acidfrom or to the sequence providing the resultant amino acid sequence hasthe same function as that presented herein, preferably being at least asbiologically active as the same. In particular, the term “homologue”covers homology with respect to structure and/or function providing theresultant amino acid sequence has the same function as that presentedherein. With respect to sequence homology, preferably there is at least75%, more preferably at least 85%, more preferably at least 90% homologyto the sequences shown herein. More preferably there is at least 95%,more preferably at least 98%, homology to the sequences shown herein.

In summation, the present invention relates to a vector comprising (a) aNS coding for a TIP and optionally (b) an NOI which encodes a POI;wherein the TIP is capable of recognising a tumour, such that in use thevector is capable of delivering the NOI and/or the POI to the tumour.

A preferred aspect of the present invention relates to a vectorcomprising (a) a NS coding for a TIP and (b) an NOI which encodes a POI;wherein the TIP is capable of recognising a tumour, such that in use thevector is capable of delivering the NOI and/or the POI to the tumour.

A further preferred aspect of the present invention relates to a vectorcomprising (a) a NS coding for a TIP and (b) an NOI which encodes a POI;wherein the TIP is capable of recognising a tumour, such that in use thevector is capable of delivering the NOI and/or the POI to the tumour;and wherein the TIP and POI are fused to each other.

This aspect of the present invention is advantageous as it allows forthe production and delivery of, for example, a fusion product thatcomprises an effector component and a targetting component.

A further preferred aspect of the present invention relates to a vectorcomprising (a) a NS coding for a TIP and (b) an NOI which encodes a POI;wherein the TIP is capable of recognising a tumour, such that in use thevector is capable of delivering the NOI and/or the POI to the tumour;wherein the TIP and POI are fused to each other; and wherein the POI iscapable of being secreted.

This aspect of the present invention is highly advantageous as itprovides a means for the in situ production of a POI by, for example, asmall number of cells for the subsequent delivery of at least a portionof the produced POI to at least one neighbouring cell. Thus, one needonly infect a small number of cells to achieve a beneficial therapeuticeffect.

Thus, alternatively expressed, the present invention provides the use ofa vector according to the present invention as an in situ productionfactory of any one or more of the NS, NOI, POI and TIP.

In addition, the present invention provides the use of a vectoraccording to the present invention when present in a cell to deliver anNOI and/or POI to a neighbouring cell.

A more preferred aspect of the present invention relates to a vectorcomprising (a) a NS coding for a TIP, (b) an NOI which encodes a POI,and (c) a nucleotide sequence that codes for a secretory entity; whereinthe TIP is capable of recognising a tumour, such that in use the vectoris capable of delivering the NOI and/or the POI to the tumour; whereinthe TIP and POI are fused to each other; and wherein the POI is capableof being secreted.

The invention will now be further described by way of examples, whichare meant to serve to assist one of ordinary skill in the art incarrying out the invention and are not intended in any way to limit thescope of the invention. Reference is made to the following Figures:

FIG. 1 b-1 d—which shows the cDNA sequence encoding 5T4Sab1. Thesequence begins with a HindIII restriction site followed by atranslation initiation signal and a signal peptide.

FIG. 1 b—which shows the cDNA sequence encoding 5T4Sab1. The sequencebegins with a HindIII restriction site followed by a translationinitiation signal and a signal peptide.

FIG. 2 a-c shows the sequence of B7-1.5T4.1

FIG. 3 shows a diagrammatic representation of two SCMs based on the B7-1co-stimulatory domain; FIG. 3 a shows the SCM B7-1.5T4.1 and FIG. 3 bshows B7- 1.5T4.2 in which the order of the co-stimulatory andtumour-binding domains are reversed. Sp=signal peptide; B7ec=extracellular domain of B7-1; V1=light chain variable domain of 5T4;Vh=heavy chain variable domain of 5T4.

FIG. 4 a-b shows the sequences of the extracellular domain of humanB7-2, including the signal peptide sequences. The mature protein beginsat amino acid 17. The B7-2derived sequences is followed by a flexiblelinker gly-gly-gly-gly-ser (SEQ ID NO: 25).

FIG. 5 shows a simple, generic diagram (not to scale) of a retroviralgenome including the elementary features of the LTRs, gag, pol, and env.

EXAMPLES Example 1 Construction of 5T4 Sab and Retroviral-VectorDelivery to Tumour

The cDNA encoding the murine 5T4 monoclonal antibody is cloned andsequenced by standard techniques (Antibody engineering: a practicalapproach ed McCafferty et al. 1996 OUP). The sequence of the variableregion of the antibody can be used to construct a variety ofimmunoglobulin-like molecules including scFvs. The coding sequence of a5T4 scFv, 5T4scFv.1, is shown in FIG. 1 a. In this molecule, the DNAsequence encodes the Vh from the mouse 5T4 monoclonal antibody followedby a 15 amino acid flexible linker and the V1 region of the mouse 5T4antibody. The flexible linker encodes 3 copies of the amino-acidsequence gly-gly-gly-gly-ser (SEQ ID NO: 25) and the DNA sequencesimilarity between the repeats has been minimise to avoid the risk ofrecombination between the repeats when plasmids containing them aregrown in E. coli.

The DNA sequence shown in FIG. 1 a can also be used to construct avariety of single-chain antibodies (Sabs) by coupling scFv-encodingsequences to a sequence encoding a Fc region to form an in-frame fusion.A Sab is constructed using a series of DNA cassettes which can beindependently varied to suit particular purposes.

Cassette 1-Translation Initiation Signal and Signal Peptide

In order to achieve correct translation initiation and secretion frommammalian cells, the following sequence is used:

(SEQ ID NO: 23) aagcttCCACCATGGGATGGAGCTGTATCATCCTCTTCTTGGTAGCAACAGCTACAGGTGTCCACTCC

This contains a convenient HindIII restriction site for cloning intoexpression vectors (lower case), the consensus translation initiationsignal for mammalian cells (ANNATGPu) and the coding sequence for asignal peptide sequence from an immunoglobulin gene.

Cassette 2-scFv

The sequence of the secreted portion of the 5T4scFv.1 is shown in FIG. 1a. This molecule can be represented as Vh-(gly4-ser) 3 linker-V1.

5T4 scFv2 consists of the 5T4 variable region sequences connected in theorder V1-flexible linker Vh. In this case the linker encodes the 20amino-acid peptide four copies of the amino acid sequencegly-gly-gly-gly-ser (SEQ ID NO: 25). A longer linker improves assemblyof the scFv when the V-region segments are in this order. (Pluckthun etat in Antibody Engineering: a practical approach, ed McCafferty et al.1996 OUP).

Cassette 3-Heavy Chain Constant Region

The sequence of a human gl constant region genomic clone is given inEllison et al. 1982 Nucl. Acids res. 10: 4071-4079. This sequencecontains constant-region introns in addition to the coding sequence.This is fused in-frame to the 3′-end of one of the scFv sequences fromCassette 2. Vectors for convenient assembly of such constructs aredescribed (Walls et al. 1993 Nucl. Acids Res. 21: 2921-2929.

A cDNA of a 5T4 Sab, designated 5T4Sab1 is shown in FIG. 1 b, containingcassettes 1, 2 and 3.

For expression of a 5T4-specific scFv or Sab in human cells, the codingsequence is inserted into the vector pCIneo (Promega) under the controlof a strong promoter and polyadenylation signal. The translationinitiation signal and immunoglobulin leader (signal peptide) sequencefrom Cassette 1 at the 5′end of the coding region ensure efficientsecretion of the scFv or Sab from mammalian cells.

For expression of an intact Ig, two separate translation cassettes areconstructed, one for the heavy chain and one for the light chain. Theseare separated by an internal ribosome-entry site (IRES) from thepicornavirus FMDV (Ramesh et al. 1996 Nucl. Acids Res. 24: 2697-2700.Alternatively, each cDNA is expressed from a separate copy of the hCMVpromoter (Ward and Bebbington 1995 In Monoclonal Antibodies ed Birch andLennox. Wiley-Liss).

For production of retrovirus capable of expressing 5T4 antibody orimmunoglobulin-like molecules with 5T4 specificity, the gene encoding a5T4-based Sab, or a dicistronic message encoding heavy and light chains,is inserted into a retroviral vector in which retroviral genomictranscripts are produced from a strong promoter such as the hCMV-MIEpromoter. A suitable plasmid is pHIT111 (Soneoka et al. 1995 Nucl. AcidsRes. 23; 628- 633) and the required gene is inserted in place of theLacZ gene using standard techniques. The resulting plasmid, pHIT-5T4.1is then transfected into the FLYRD18 or FLYA13 packaging cell lines(Cosset et al. 1995 J. Virol. 69; 7430-7436) and transfectants selectedfor resistance to G418 at 1 mg/ml. G418-resistant packaging cellsproduce high titres of recombinant retrovirus capable of infecting humancells. The virus preparation is then used to infect human cancer cellsand can be injected into tumours in vivo. The 5T4 Sab is then expressedand secreted from the tumour cells.

In pHIT111, the MoMLV LTR promoter-enhancer is used for expression ofthe therapeutic gene in the target cell. The vector can also be modifiedso that the therapeutic gene is transcribed from an internalpromoter-enhancer such as one which is active predominantly in thetumour cells or one which contains a hypoxia regulated element. Asuitable promoter is a truncated HSV TK promoter with 3 copies of themouse PGK HRE (Firth et al. 1994 Proc. Natl. Acad. Sci. 91: 6496-6500).

Example 2 Transfection of Macrophages/Monocytes with an ExpressionVector Encoding TBP

Peripheral blood mononuclear cells are isolated from human peripheralblood at laboratory scale by standard techniques procedures (Sandlie andMichaelsen 1996 In Antibody engineering: a practical approach. EdMcCafferty et al. Chapter 9) and at large scale by elutriation (e.g.Ceprate from CellPro). Adherent cells (essentially monocytes) areenriched by adherence to plastic overnight and cells can be allowed todifferentiate along the macrophage differentiation pathway by culturingadherent cells for 1-3 weeks.

Monocytes and macrophages are transfected with an expression vectorcapable of expressing TBP in human cells. For constitutive high levelexpression, the TBP is expressed in a vector which utilises the hCMV-MIEpromoter-enhancer, pCI (Promega). For hypoxia-induced expression, thehCMV promoter is replaced by a promoter containing at least one HRE. Asuitable promoter is a truncated HSV TK promoter with 3 copies of themouse PGK HRE (Firth et al. 1994 Proc. Natl. Acad. Sci. 91: 6496-6500).

A variety of transfection methods can be used to introduce vectors intomonocytes and macrophages, including particle-mediated DNA delivery(biolistics), electroporation, cationic agent-mediated transfection(e.g. using Superfect, Qiagen). Each of these methods is carried outaccording to the manufacturer's instructions, taking into account theparameters to be varied to achieve optimal results as specified by theindividual manufacturer. Alternatively, viral vectors may be used suchas defective Adenovirus vectors (Microbix Inc. or QuantumBiotechnologies Inc.).

Example 3 Assay for ADCC Mediated by Macrophages

Cells from primary human tumours or tumour cell lines which have beentransduced with retrovirus expressing TBP are mixed with autologous orheterologous human macrophages, prepared as described in Example 2, foranalysis of ADCC activity mediated by the TBP. Alternatively,macrophages engineered to produce TBP as described in Example 2 can beused to direct ADCC on non-transduced tumour cells.

The assay is carried out according to standard procedures (Sandlie andMichaelsen 1996 In Antibody engineering: a practical approach. EdMcCafferty et al. Chapter 9) with appropriate modifications. Briefly,the effector cells (macrophages or freshly isolated monocytes) aresuspended at 3×10⁶ cells/ml in the appropriate tissue culture medium(DMEM/Hepes, obtained from Life Technologies, containing 1% Foetal CalfSerum). 3×10⁵ tumour target cells, labelled with ⁵¹Cr are placed in eachwell of a round-bottomed microtitre plate in 0.1 ml culture medium.(Note the culture medium can include spent medium from the cellsproducing the TBP). 50 ml effector cells are added to the wells, theplate is centrifuged at 300 g for 2 min and incubated at 37° C. forvarying periods (e.g. 4 h) in a tissue culture incubator. Thesupernatant is then harvested by centrifugation and counted in a gammacounter. Results are expressed as percent lysis relative to totalchromium release from an equivalent sample of target cells lysed with0.1% Tween-20. The effector: target cell ratio can be varied in theassay to produce a titration curve.

For the prior stimulation of macrophage differentiation or priming,cytokines are added to the cultures. IFNg (Sigma) is added at between100 and 5000 U/ml. CSF-1 or GM-CSF (Santa Cruz Biotechnology) can alsobe added at appropriate concentrations.

Example 4 Analysis of Efficacy in Animal Models

Human tumour-derived cell lines and tissues are cultured in vivo ingenetically immunodeficient, “nude” mice according to well establishedtechniques (see for example Strobel et al. 1997 Cancer Res. 57:1228-1232; McLeod et al. 1997 Pancreas 14: 237- 248). Syngeneic mousemodels, in which a syngeneic tumour line is introduced into animmunocompetent mouse strain may also be used. These serve as suitableanimal models for evaluating gene delivery systems of the invention.Vectors or engineered cells are administered systemically or directlyinto the tumour and tumour growth is monitored in treated and untreatedanimals. This system is used to define the effective dose range of thetreatments of the invention and the most appropriate route ofadministration.

Example 5 Construction of B7-scFv Fusion Proteins

The extracellular domain of B7-1 is defined by amino-acid residues 1-215of the native human B7-1 protein. This sequence, together with itssignal peptide-encoding sequence, is used to construct secreted fusionproteins which also contain the scFv derived from the 5T4 monoclonalantibody. The sequence of the 5T4 scFv is given in FIG. 1 a.

A DNA coding sequence is constructed using standard molecular biologytechniques which encodes a fusion protein in which the N-terminus of the5T4 scFv is fused after amino acid 215 of human B7-1. The sequence ofthis coding sequence, B7-1.5T4.1, is shown in FIG. 2. The fusion proteincontains a flexible (gly-gly-gly-gly-ser, SEQ ID NO: 25)) spacer betweenthe B7-1 and 5T4 scFv sequences. The introduction of a convenient BamHIrestriction site at the end of the linker insertion (beginning atnucleotide 733) also allows for further linkers to be screened foroptimal expression of bi-functional fusion protein. FIG. 3 indicates thefusion protein in diagrammatic form. It is similarly possible toconstruct B7-1.5T4.2 (FIG. 3 b) in which the scFv is N-terminal and theB7 extracellular domain is C-terminal. In this case only the codingsequence of the mature B7-1 (without signal peptide) is required. Asignal peptide such as an immunoglobulin leader sequence is added to theN-terminus of the scFv in this instance.

For fusion proteins which use the co-stimulatory extracellular domain ofB7-2, the signal peptide and extracellular domain of B7-2 is used inplace of B7-1 sequences. FIG. 4 shows the coding sequence of the SCMB7-2.5T4. lco-stimulatory domain. It encodes the first 225 amino acidsof human B7-2, preceded by its signal peptide, and a flexible linkergly-gly-gly-gly-ser, SEQ ID NO: 25. The BamHI site at the end of thissequence can be used to insert the domain upstream of the 5T4scFv. 1(see FIG. 3). The sequence includes the B7-2 signal peptide which canserve to allow secretion of this fusion protein in which the B7-2 domainis at the N-terminus of the fusion protein.

Each engineered cDNA is inserted into the mammalian expression vectorpCI to allow expression in mammalian tissue culture cells. For thispurpose, a linker sequence is added to the 5′-end of the coding sequencewhich introduces a convenient restriction site for insertion into thepolylinker of pCI and adds the translation initiation signal CCACCimmediately adjacent to the first ATG codon. Constructs in pCI aretransfected into a suitable mammalian host cell line such as COS-1 toconfirm secretion of the SCM. The transcription cassette from pCI or anappropriate segment of the transcription cassette is subsequentlysub-cloned into the expression vector to be used as the gene deliverysystem for therapeutic use.

Example 6 Transfection of Macrophages/Monocytes with an ExpressionVector Encoding an SCM

Peripheral blood mononuclear cells are isolated from human peripheralblood at laboratory scale by standard techniques procedures (Sandlie andMichaelsen 1996 In Antibody engineering: a practical approach. EdMcCafferty et al. Chapter 9) and at large scale by elutriation (e.g.Ceprate from CellPro). Adherent cells (essentially monocytes) areenriched by adherence to plastic overnight and cells can be allowed todifferentiate along the macrophage differentiation pathway by culturingadherent cells for 1-3 weeks.

Monocytes and macrophages are transfected with an expression vectorcapable of expressing SCM in human cells. For constitutive high levelexpression, the SCM is expressed in a vector which utilises the hCMV-MIEpromoter-enhancer, pCI (Promega). For hypoxia-induced expression, thehCMV promoter is replaced by a promoter containing at least one HRE. Asuitable promoter is a truncated HSV TK promoter with 3 copies of themouse PGK HRE (Firth et al. 1994 Proc. Natl. Acad. Sci. 91: 6496-6500).

A variety of transfection methods can be used to introduce vectors intomonocytes and macrophages, including particle-mediated DNA delivery(biolistics), electroporation, cationic agent-mediated transfection(e.g. using Superfect, Qiagen). Each of these methods is carried outaccording to the manufacturer's instructions, taking into account theparameters to be varied to achieve optimal results as specified by theindividual manufacturer. Alternatively, viral vectors may be used suchas defective Adenovirus vectors (Microbix Inc. or QuantumBiotechnologies Inc.).

Example 7 Analysis of SCM Binding to CTLA-4 and 5T4-Antigen ExpressingCells

The B7-1 or B7-2 domains are expected to bind specifically to CD28 andCTLA-4 present on human T-cells. Binding to T-cells or Chinese hamsterovary cells transfected with human CTLA-4 or CD28 is determined usingFACS analysis as follows. 5×10⁵ CTLA-4 expressing target cells orequivalent cells lacking CTLA-4 (untransfected CHO cells) are incubatedwith 0.1 ml culture supernatant from COS-1 cells transiently transfectedwith SCM genes for 1 h at 4° C. The cells are washed and incubate with 1mg monoclonal antibody specific for the B7 domain (e.g. Mab 9E10)followed by FITC-labelled goat anti-mouse IgG (Pharmingen) and analysisby FACS.

Binding of scFv to 5T4-antigen is similarly assessed using target cellsexpressing 5T4- antigen (5T4-transfected A9 cells) or control cells(A9).

Example 8 Analysis of Co-Stimulatory Activity

An established mouse cell line of Balb/c origin such as HC11 cells istransfected with the cDNA encoding human 5T4-antigen (Myers et al. 1994J. Biol. Chem. 269; 9319-9324) inserted in the expression vector pCIneo.

Splenic T-cells from Balb/c mice are isolated by standard procedures(Johnstone and Thorpe 1996 In Immunochemistry in Practice. Blackwell.Chapter 4). T-cells are pre-stimulated by incubation for 1-2 days inmedium containing 10 ng/ml PMA (Sigma) and 100 U/ml human IL-2(Boehringer Mannheim). HC11-5T4 cells are incubated at 10⁴ cells /wellof a 96-well tissue culture tray for 2 h with up to 0.1 ml supernatantfrom COS cells transfected with SCM gene. Up to 10⁵ pre-stimulatedT-cells are added to each well, the cells are pulsed with 0.25 mCi/well³H-thymidine and incorporation of ³H-thymidine is measured using aliquid scintillation counter after 24 h.

Incorporation of ³H-thymidine is anticipated to be enhanced by thepresence of SCM.

Example 9 Analysis of Co-Stimulation in Animal Models

HC11 cells transfected with the human 5T4-antigen gene (Example 4) aregrown as tumours in Balb/c mice. SCM genes B7-1.5T4.1 or B7-2.5T4.1 or acombination of both genes are introduced into the tumour cells prior toimplantation and the growth of the tumours and the growth of controltumours which do not express SCM genes in vivo are monitored.

It is believed that the expression of SCM genes lead to significantreduction in tumour growth.

Example 10 Construction of a B7-1/ScFv, Specific for Human 5T4, FusionProtein

Standard molecular biology techniques are used to construct a fusionprotein consisting of the leader sequence and extracellular domain ofB7-1, fused via a flexible linker to the VH and VL of the murine Mab 5T4specific to human 5T4.

The flexible linker, used to join the extracellular domain of B7.1 andthe ScFv, was constructed by annealing two homologous oligonucleotideswith engineered 5′ Sma I and 3′ Spe I sites-using oligonucleotides

upper

(SEQ ID NO: 24) 5′ GGG GGT GGT GGG AGC GGT GGT GGC GGC AGT GGCGGC GGC GGA A 3′and lower

(SEQ ID NO: 8) 5′ CTA GTT CCG CCG CCG CCA CTG CCG CCA CCA CCGCTC CCA CCA CCC CC 3′

The linker is cloned into pBluescript (Stratagene) via Sma I and Spe Ito produce pLINK. The signal peptide (sp) and extracellular domain ofmurine B7.1 were ampified by PCR from pLK444-mB7.1 (supplied by R.Germain NIH, USA) via primers that introduce 5′ EcoRI and 3′Sma Isites-primers forward

(SEQ ID NO: 9) 5′ C TCG AAT TCC ACC ATG GCT TGC AAT TGT CAG TTG ATG C 3′reverse

(SEQ ID NO: 10) 5′ CTC CCC GGG CTT GCT ATC AGG AGG GTC TTC 3′

The B7.1 PCR product was cloned into pLINK via Eco RI and Sma I to formpBS/B7Link.

The V_(H) and V_(L) Of the 5T4 specific ScFv was amplified viaprimers-forward primer

(SEQ ID NO: 11) 5′ CTC ACT AGT GAG GTC CAG CTT CAG CAG TC 3′reverse primer

(SEQ ID NO: 12) 5′ CTC GCG GCC GCT TAC CGT TTG ATT TCC AGC TTGGTG CCT CCA CC 3′

That introduce 5′ Spe I and 3′ Not I sites from pHEN1-5T4 ScFv.PBS/B7Link was digested with Spe I and Not I and ligated with the ScFvto form OBM 233 consisting of the sequence shown as SEQ ID No. 5: B7Link scFv sequence.

This fusion can be used to construct a recombinant vector e.g.retrovirus, Lentivirus, adenovirus, poxvirus, vaccina virus,baculovirus. Such vectors can be used to inject patient tumoursdirectly. To deliver the fusion protein to tumour cells the recombinantvector is used to transduce macrophages/monocytes/CD34+ cells ex vivobefore injection back into patients. These cells will traffic totumours. The ScFv will bind to a specific tumour antigen expressed onthe surface of tumour cells e.g. 5T4 (Myers et al 1994 JBC). B7 is foundon the surface of professional antigen presenting cells e.g.macrophages, dendritic cells and B cells. It interacts with it ligandsCD28 and CTL-A4 located on CD4 and CD8 cells. The simultaneousinteraction of B7-CD28/CTL-A4 and MHC-peptide/T cell receptor leads to apronounced increase in IL-2 which promotes CD8 (cytotoxic T cell)expansion (Linsley P S, Brady W, Grosmaire L, Aruffo A, Damle N K,Ledbetter J A J Exp Med 1991 Mar 1; 173 (3): 721-730 Binding of the Bcell activation antigen B7 to CD28 costimulates T cell proliferation andI1-2 mRNA accumulation.) Tumour cells that have been B7 tranfected withB7 have been shown retardation in animal models (Townsend S E, Allison JP Science 1993 15; 259 (5093): 368-370).

Example 11 Transient Expression and Purification of B7-1/ScFv and LScFv

For transient expression of B7-1/ScFv the human CMV expression plasmidpCIneo (Promega) was used. B7/ScFv was excised from OBM 233 by digestionwith EcoR I/Not I and cloned into pCIneo that was previously digestedwith EcoRI/Not I. Transient expression of recombinant protein is made bytransfection of 293T cells with the relevant plasmid using calciumphosphate (Profectin, Promega). Conditions used were similar to thoserecommended by the manufacturer. To reduce bovine serum contaminationserum free optimem media (Gibco BRL). After 36-48 hours transfectionsupernatants were harvested and spun through a Centriprep (Amicon, Glos.UK) 10 filter (all proteins larger than 10 kDa arepurified/concentrated) and a Centricon (Amicon) 10 filter. Supernatantsare concentrated approximately 30 fold.

For B7-1 to be biologically functional it must be able to displaybinding with one of it's natural ligands either CTLA-4 or CD28 found onthe surface of specific populations of T cells (e.g CD4+). Thebiological activity B7-1/ScFv fusion protein was analysed forsimultaneous interaction with its natural ligand CTLA-4 (in the form ofCTLA4-Ig supplied by Ancell, Minn., USA) and A9 cells expressing human5T4. Briefly: approximately 5×10⁵ A9-h5T4 cells were incubated with 100ul of either B7.1/ScFv or LScFv supernatant in a U bottom 96 well plateat 4° C. for 1 hour. After washing cells were incubated with CTLA4-Ig(Ancell) for 1 hour. After washing, bound CTLA4-Ig was detected using anFITC conjugated anti-mouse Ig (Dako).

Results show obvious binding of CTLA-Ig with the B7-1 extracellulardomain, bound via the ScFv, to the surface of human 5T4 positive A9cells. The lack of binding activity with 5T4 negative A9 cells furtherillustrates that the interaction of B7 with CTLA4-Ig and ScFv with 5T4are specific.

Example 12 ScFv-IgG Fusion Example

Construction of ScFv-IgG

The sequence encoding a translation initiation sequence and the humanimmunoglobulin kappa light chain signal peptide is synthesised as twocomplementary single stranded oligonucleotides which when annealed alsocontain an internal Xho I site at the 5′ end and in addition leave a XbaI compatible 5′ overhang and a Pst I compatible 3′ overhang

(SEQ ID NO: 13) ctagactcgagCCACC ATG GGA TGG AGC TGT ATC ATC CTCTTC TTG GTA GCA ACA GCT ACA GGT GTC CAC TCC GAG GTC CAG ctgca and(SEQ ID NO: 14) g CTG GAC CTC GGA GTG GAC ACC TGT AGC TGT TGC TACCAA GAA GAG GAT GAT ACA GCT CCA TCC CAT GGTGGctcg agt

This is then cloned into pBluescript II (Stratagene) restricted with XbaI and Pst I to create pBSII/Leader.

The 5T4 scFv is amplified by PCR from pHEN1 using oligonucleotides whichincorporate a Pst I site at the 5′end of the product and a Hind III atthe 3′end

GTC CAG CTG CAG CAG TCT GG (SEQ ID NO: 15) andCG TTT GAT TTC AAG CTT GGT GC (SEQ ID NO: 16)

This is then restricted with those enzymes and inserted intopBSII/Leader restricted with the same enzymes, creatingpBSII/Leader/scFv.

The HIgG1 constant region is amplified by PCR from the cloned gene usingoligonucleotides which incorporate a Hind III site at the 5′end and aXho I site at the 3′ end

(SEQ ID NO: 17) gcgc AAG CTT gaa atc aaa cgg GCC TCC ACC AAG GGC CCA and(SEQ ID NO: 19) gcgc ctcgag TCA TTT ACC CGG AGA CAG GG

This is then restricted with those enzymes and inserted intopBSII/Leader/scFv restricted with the same enzymes, creatingpBSII/Leader/scFv/HG1. The sequence for this construct is shown in theFigures.

This fusion can be used to construct a recombinant vector e.g.retrovirus, Lentivirus, adenovirus, poxvirus, vaccinia virus,baculovirus. Such vectors can be used to inject patient tumoursdirectly. To deliver the fusion protein to tumour cells the recombinantvector is used to transduce macrophages/monocytes/CD34+ cells ex vivobefore injection back into patients. These cells will traffic totumours. The ScFv will bind to a specific tumour antigen expressed onthe surface of tumour cells e.g. 5T4 (Myers et al 1994 JBC). Bound IgGwill promote specific tumour destruction via a collection of mechanismscollectively known as antibody dependent cellular cytotoxicity (Munn etal Can res 1991 ibid, Primus et al 1993 Cancer Res ibid).

Example 13 Construction of ScFv-IgEl (Human IgEl Heavy Constant Region)

A similar fusion construct of 5T4 scFv-human IgE constant heavy chain ismade consisting of the sequence shown as SEQ ID No. 6.

This fusion construct is made by amplifying the human IgEl constantheavy region by PCR cDNA derived from human B-cells RNA by RT andsubsequently using oligonucleotides which incorporate a Hind III site atthe 5′ end and a Xho I site at the 3′end

(SEQ ID NO: 19) gcgc AAG CTT gaa atc aaa cgg GCC TCC ACA CAG AGC CCA and(SEQ ID NO: 20) gcgc ctcgag TCA TTT ACC GGG ATT TAC AGA

This is then restricted with those enzymes and inserted intopBSII/Leader/scFv restricted with the same enzymes, creatingpBSII/Leader/scFv/HE1.

As described above the ScFv-IgE construct can be incorporated into arecombinant viral vector for use in gene therapy of cancer e.g. injectpatient tissue directly or to transduce patient derivedmacrophages/moncytes/CD34+ cells ex vivo. The fusion protein will besecreted and will bind to tumour cells bearing the antigen that the ScFvis specific for. Binding of IgE to tumour cells should promote a stronghistamine response via activation of mast cells. This will lead to astrong inflammatory response and destruction tumour cells as is reportedfor IgE cytotoxic destruction of parasites e.g. helminth larvae (CapronM 1988 Eosinophils in diseases: receptors and mediators. In progress inallergy and clinical immunology (Proc. 13^(th) Int. Congress of Allergyand Clinical Immunology) Hogrefe & Huber Toronto p 6). Such inflammationand tumour destruction should initiate the recruitment of other immuneeffector cells. Past reports indicate that treatment with an MMTVantigen specific IgE Mab leads to protection from a tumour expressingMMTV antigen (Nagy E Istanvan B, Sehon A H 1991 Cancer Immunol.Immunotherapy vol 34: 63-69).

Example 14 Construction of B7/EGF

B7-EGF Synthetic Gene.

A fusion construct of B7-EGF is made by inserting a PCR productamplified from the region of the gene encoding the mature EGF peptide(see accession number X04571) into pBS/B7 Link. This construct has thesequence shown as SEQ ID No. 7.

Using cDNA derived by RT of RNA isolated from a cell line such the 293human kidney line (ATCC: CRL1573), the DNA is amplified by PCR usingoligonucleotides containing a Spe I restriction enzyme site at theN-terminus and a stop codon and a Not I site at the C-terminus

(SEQ ID NO: 21) GG ACT AGT AAT AGT GAC TCT GAA TGT CCC and(SEQ ID NO: 22) ATT AGC GGC CGC TTA GCG CAG TTC CCA CCA CTT C

The resulting product is digested with those enzymes and ligated topBS/B7 Link which has been restricted with the same enzymes creatingpBS/B7 Link EGF. The B7 Link EGF cassette is then excised with Eco RIand Not I and inserted into a derivative of pHIT111 (Soneoka et al,1995, Nucl Acid Res 23; 628) which no longer carries the LacZ gene.

An alternative to using ScfV is to use growth factors that have a highaffinity to their corresponding receptor e.g. epidermal growth factorwhich binds to several receptors including erb-2 which is highlyassociated with tumourgenesis.

As described above the fusion construct can be incorporated into arecombinant viral vector for use in gene therapy e.g. inject patienttissue directly or to transduce patient derivedmacrophages/moncytes/CD34+ cells ex vivo. The fusion protein will besecreted and will bind to tumour cells bearing the erb-2 antigen.

Epidermal growth factor (EGF) will bind to its ligand erb-2 (an EGFreceptor) thus obviating the requirement of a ScFv. Erb-2 is highlyassociated with tumour cells (Hynes N E Semin Cancer Biol 1993 Feb. ; 4(1): 19-26, Amplification and over expression of the erbB-2 gene inhuman tumors: its involvement in tumor development, significance as apronostic factor, and potential as a target for cancer therapy). B7 isfound on the surface of professional antigen presenting cells e.g.macrophages, dendritic cells and B cells. It interacts with it ligandsCD28 and CTL-A4 located on CD4 and CD8 cells. The simultaneousinteraction of B7-CD28/CTL-A4 and MHC-peptide/T cell receptor leads tomassive increase in IL-2 which promotes CD8 (cytotoxic T cell) expansion(Linsley P S, Brady W, Grosmaire L, Aruffo A, Damle N K, Ledbetter J A JExp Med 1991 Mar. 1 ; 173 (3): 721-730 Binding of the B cell activationantigen B7 to CD28 costimulates T cell proliferation and interleukin 2mRNA accumulation.) Tumour cells that have been B7 transfected with B7have shown retardation in animal models (Townsend S E, Allison J PScience 1993 15; 259 (5093): 368-370 Tumor rejection after directcostimulation of CD8+ T cells by B7-transfected melanoma cells). It ishas been reported that B7 will enhance the CTL response to tumourantigens specific to tumour cells thus leading to the destruction of allsuch cells.

Example 15 Production of Cell Lines Expressing Fusion Constructs

The ScFv-IgG gene was excised from pBSII/L/ScFv/hIgG1 by Xho Idigestion, and cloned into pLXSN via the Xho I site, to makepLXSN/ScFv-IgG, such that after chromosomal integration it is undertranscriptional control of the LTR. Virus was made in the human kidneycell line 293T by co-transfecting plasmids containing the MLV gap-polgenes (pCIEGPPD) and the VSV G envelope (pRV67) using the triple plasmidHIT system (Landau & Littman 1992 J Virol 66 5110, Soneoka Y et al 1995NAR 23: 628-633). Virus is harvested after 48 hours and used totransduce BHK-21 cells (ATCC#CCL-10). Approximately 24 hourspost-transduction, transduced cells are selected by the addition of 1mg/ml G418 (Gibco BRL) to culture medium. The supernatant from positivecolonies was harvested and concentrated by centrifugation through aCentriprep (Amicon, Glos. UK) 10 filter (all proteins larger than 10 kDaare purified/concentrated) and a Centricon (Amicon) 10 filter.Supernatants were concentrated approximately 30 fold.

Other fusion proteins are cloned into pLXSN via the Xho I site andexpressed and concentrated using a similar protocol.

FACS analysis of fusion protein binding with cells expressing specificligand To determine if the ScFv-IgG fusion protein is specific for itsantigen, human 5T4, FACS analysis of a human bladder carcinoma tumourline (EJ) or a stable murine cell line expressing h5T4, A9-h5T4 (Myerset al 1994 JBC) and a 5T4 negative line A9-neo was carried out.Approximately 5×10⁵ A9 or EJ cells, in a round bottom 96 well plate(Falcon) were incubated with 100 ul of a 1:5 dilution of concentratedsupernatant (as described above) for 1 hour at 4° C. After washing,bound protein is detected using an anti human IgG/FITC conjugatedantibody (Dako). Cells were analysed on a Becton Dickinson FACS machine.FACS results show that there is at least a 1 log shift in fluorescenceactivity in those 5T4 positive cells treated with the ScFv-IgG constructcompared to the negative control construct consisting of the ScFvprotein alone. A9 neo FACS shows that there is no non-specific bindingof the ScFv component of the fusion protein.

FACS analysis of ScFv-IgE is carried out similar to above except thatanti-human IgE-FITC (Dako) is used to detect binding of the fusionprotein.

The B7/EGF fusion protein is analysed for binding using FACS andHC11-erb-2 positive cells (Hynes et al 1990). CTLA4-Ig (Ancell, USA) isused to analyse the bioactivity of the B7 component of the bound fusionprotein. Anti-mouse IgG-FITC is used to show CTLA- 4 binding.

SUMMARY

The present invention therefore provides a means for delivering, forexample, therapeutic compounds to a tumour site.

All publications mentioned in the above specification are hereinincorporated by reference. Various modifications and variations of thedescribed methods and system of the invention will be apparent to thoseskilled in the art without departing from the scope and spirit of theinvention. Although the invention has been described in connection withspecific preferred embodiments, it should be understood that theinvention as claimed should not be unduly limited to such specificembodiments. Indeed, various modifications of the described modes forcarrying out the invention which are obvious to those skilled inmolecular biology or related fields are intended to be within the scopeof the following claims.

1. A vector comprising a nucleotide sequence coding for a proteinconsisting essentially of an antibody that binds 5T4, said antibodycomprising 5T4 complementarity determining regions (CDRs) of the 5T4ScFv antibody amino acid sequence encoded by SEQ ID NO:1 or CDRs of the5T4 Sab antibody amino acid sequence encoded by SEQ ID NO:2.
 2. Thevector according to claim 1, wherein the antibody is a 5T4 Fab, 5T4 Fvor 5T4 scFc fragment.
 3. The vector according to claim 1, wherein theantibody is a humanized antibody.
 4. A vector comprising a nucleotidesequence coding for a protein consisting essentially of an antibody thatbinds 5T4, said antibody comprising 5T4 complementarity determiningregions (CDRs) having the amino acid sequence of KASQSVSNDVA (SEQ IDNO:26), YTSSRYA (SEQ ID NO:27), QQDYNSPPT (SEQ ID NO:28), GYYMH (SEQ IDNO:29), RINPNNGVTLYNQKFKD (SEQ ID NO:30), and STMITNYVMDY (SEQ IDNO:31).
 5. The vector according to claim 4, wherein the antibody is a5T4 Fab, 5T4 Fv or 5T4 scFc fragment.
 6. The vector according to claim4, wherein the antibody is a humanized antibody.